Inhibition of bacterial biofilms with imidazole derivatives

ABSTRACT

Disclosure is provided for imidazole derivative compounds that prevent, remove and/or inhibit the formation of biofilms, compositions comprising these compounds, devices comprising these compounds, and methods of using the same.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application Ser. No. 60/886,789, filed Jan. 26, 2007,the disclosure of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods useful forcontrolling biofilms.

BACKGROUND OF THE INVENTION

Biofilms are complex communities of microorganisms that are commonlyfound on a variety of substrates or surfaces that are moist or submerged(Musk et al., Curr. Med. Chem., 2006, 13, 2163; Donlan et al., Clin.Microbiol. Rev., 2002, 15, 167). Though primarily populated by bacteria,biofilms can also contain many different individual types ofmicroorganisms, e.g., bacteria, archaea, protozoa and algae. Theformation of biofilms can be thought of as a developmental process inwhich a few free-swimming (planktonic) bacteria adhere to a solidsurface and, in response to appropriate signals, initiate the formationof a complex sessile microcolony existing as a community of bacteria andother organisms. Bacteria within biofilms are usually embedded within amatrix, which can consist of protein, polysaccharide, nucleic acids, orcombinations of these macromolecules. The matrix is a critical featureof the biofilm that protects the inhabiting organisms from antiseptics,microbicides, and host cells. It has been estimated that bacteria withinbiofilms are upwards of 1,000-fold more resistant to conventionalantibiotics (Rasmussen et al., Int. J. Med. Microbiol., 2006, 296, 149).

Biofilms play a significant role in infectious disease. It is estimatedthat biofilms account for between 50-80% of microbial infections in thebody, and that the cost of these infections exceeds $1 billion annually.For example, persistent infections of indwelling medical devices remaina serious problem for patients, because eradication of these infectionsis virtually impossible. A few diseases in which biofilms have beenimplicated include endocarditis, otitis media, chronic prostatitis,periodontal disease, chronic urinary tract infections, and cysticfibrosis. The persistence of biofilm populations is linked to theirinherent insensitivity to antiseptics, antibiotics, and otherantimicrobial compounds or host cells.

Cystic fibrosis (CF), with 7 million asymptomatic heterozygous carriers,is one of the most common genetic diseases in the United States. Despitesignificant progress in the management of the symptoms of CF, virtuallyall CF patients succumb to chronic pulmonary infections. For reasonsthat are not entirely clear, the airways of CF patients are particularlysusceptible to bacterial colonization. CF patients typically becomeinfected with Staphylococcus aureus, Streptococcus pneumoniae,Haemophilus influenzae, Burkholderia cepacia complex, and nonmucoidPseudomonas aeruginosa. However, as the patients age, Pseudomonaaeruginosa becomes the predominant pulmonary pathogen, present in up to85% of cultures from patients with advanced disease. Once colonized byPseudomonas aeruginosa, the organism persists for many years or decadesand is never eradicated. This persistence of Pseudomonas aeruginosa hasbeen linked to its ability to form biofilms. Complications arising fromPseudomonas aeruginosa infections are the leading cause of death amongCF patents.

Deleterious effects of biofilms are also found in non-medical settings.For example, biofilms are a major problem in the shipping industry.Biofilms form on and promote the corrosion of ship hulls and alsoincrease the roughness of the hulls, increasing the drag on the shipsand thereby increasing fuel costs. The biofilms can also promote theattachment of larger living structures, such as barnacles, to the hull.Fuel can account for half of the cost of marine shipping, and the lossin fuel efficiency due to biofilm formation is substantial. One methodof controlling biofilms is to simply scrape the films off of the hulls.However, this method is costly and time-consuming, and can promote thespread of troublesome non-native species in shipping waters. Anothermethod involves the use of antifouling coatings containing tin. However,tin-based coatings are now disfavored due to toxicity concerns.

Given the breadth of detrimental effects caused by bacterial biofilms,there has been an effort to develop small molecules that will inhibittheir formation (Musk et al., Curr. Med. Chem., 2006, 13, 2163). Theunderlying principle is that if bacteria can be maintained in theplanktonic state, they will either not attach to a target surface and/orthey can be killed by a lower dose of microbicide.

Despite the extent of biofilm driven problems, examples of structuralscaffolds that inhibit biofilm formation are rare (Musk et al., Curr.Med. Chem., 2006, 13, 2163). The few known examples include thehomoserine lactones (Geske et al., J. Am. Chem. Soc., 2005, 127, 12762),which are naturally-occurring bacterial signaling molecules thatbacteria use in quorum sensing (Dong et al., J. Microbiol., 2005, 43,101; Nealson et al., J. Bacteriol., 1970, 104, 313), brominatedfuranones isolated from the macroalga Delisea pulchra (Hentzer et al.,Microbiology-Sgm, 2002, 148, 87), and ursene triterpenes from the plantDiospyros dendo (Hu et al., J. Nat. Prod., 2006, 69, 118). While thefocus has predominantly been on designing small molecules that inhibitthe formation of biofilms, one of the more significant challenges is thedevelopment of a small molecule that disperses pre-formed biofilms. Noneof the small molecules noted above have been previously reported todisperse an existing biofilm.

In addition, bacteria have an unparalleled ability to overcome foreignchemical insult. For example, resistance to vancomycin, “the antibioticof last resort,” has become more prevalent, and strains ofvancomycin-resistant Staphylococcus aureus have become a serious healthrisk. It has been predicted that it is simply a matter of time beforedifferent bacterial strains develop vancomycin resistance, and thesafety net that vancomycin has provided for decades in antibiotictherapy will no longer be available. Therefore, the identification ofchemical architectures useful to inhibit biofilm development is needed.

Because of their natural resistance to antibiotics, phagocytic cells,and other biocides, biofilms are difficult, if not impossible, toeradicate. Therefore, the identification of compounds that controlbiofilm formation is of critical need.

SUMMARY OF THE INVENTION

Provided herein are compounds of Formula (I):

wherein:

R¹ and R² and R³ are each independently selected from the groupconsisting of: H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone,sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain,amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

Also provided are compounds of Formula (I)(a):

wherein:

R³ is selected from the group consisting of: H, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy,amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

Further provided are compounds of Formula (II):

wherein:

R⁸ is selected from the group consisting of: H, amino, hydroxy, andthiol; and

R⁹ and R¹⁰ are each independently selected from the group consisting of:H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo,aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo,oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

Also provided are compounds of Formula (II)(b):

wherein:

R¹¹ and R¹² are each independently selected from the group consistingof: H, hydroxy, acyl, alkyl alkenyl, alkynyl, cycloalkyl, heterocyclo,aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo,oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide;

or a pharmaceutically acceptable salt or prodrug thereof Each group canbe optionally substituted.

Also provided are compounds of Formula (II)(c):

wherein:

R¹³ and R¹⁴ are each independently selected from the group consistingof. H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo,aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo,oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide;

or a pharmaceutically acceptable salt or prodrug thereof Each group canbe optionally substituted.

Further provided are compounds of Formula (III):

wherein:

R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²² are each independentlyselected from the group consisting of: H, hydroxy, acyl, alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino,amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy,amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

Provided are compounds of Formula (IV):

wherein:

R²³ is selected from the group consisting of: H, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

Also provided are compounds of Formula (V) and Formula (VI):

These formulas are also optionally substituted.

Also provided are compounds of Formula (X):

wherein:

R¹ and R² and R³ are each independently selected from the groupconsisting of: H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone,sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain,amino acid and peptide; and

A and B are each independently selected from N, S and O.

Further provided are compounds of Formula (X)(I)(a):

wherein R⁵ is an alkyl, alkenyl or alkynyl having an amide groupsubstituted thereon;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Also provided are compounds of Formula (X)(I)(a)(1):

wherein:

n is 1 to 10 carbons, saturated or unsaturated; and

R⁶ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Provided are compounds of Formula (X)(I)(a)(2):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and

R⁷ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Also provided are compounds of Formula (X)(I)(a)(2)(A):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and X, Y and Z are each independently selected H, halo,hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Provided are compounds of Formula (X)(I)(b):

wherein R⁸ is an alkyl, alkenyl or alkynyl having an amide groupsubstituted thereon;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Also provided are compounds of Formula (X)(I)(b)(1):

wherein:

n is 1 to 10 carbons, saturated or unsaturated; and

R⁹ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Further provided are compounds of Formula (X)(I)(b)(2):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and

R¹⁰ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Also provided are compounds of Formula (X)(I)(b)(2)(A):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and X, Y and Z are each independently selected H, halo,hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Provided are compounds of Formula (X)(I)(c):

wherein R¹¹ is an alkyl, alkenyl or alkynyl having an amide groupsubstituted thereon;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Also provided are compounds of Formula (X)(I)(c)(1):

wherein:

n is 1 to 10 carbons, saturated or unsaturated; and

R¹² is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Further provided are compounds of Formula (X)(I)(a)(2):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and

R¹³ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Also provided are compounds of Formula (X)(I)(c)(2)(A):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and X, Y and Z are each independently selected H, halo,hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Biofilm preventing, removing or inhibiting compositions are provided,which include a carrier and an effective amount of a compound disclosedherein. In some embodiments, the composition is a dentifrice compositionthat promotes dental hygiene by preventing, reducing, inhibiting orremoving a biofilm. In some embodiments, the dentifrice compositioncomprises a toothpaste, mouthwash, chewing gum, dental floss, or dentalcream.

Compositions are also provided that include a compound disclosed hereinin a pharmaceutically acceptable carrier.

Compositions are further provided that include a compound disclosedherein covalently coupled to a substrate. In some embodiments, thesubstrate includes a polymeric material. In some embodiments, thesubstrate includes a solid support. In some embodiments, the substrateincludes a drainpipe, glaze ceramic, porcelain, glass, metal, wood,chrome, plastic, vinyl, and Formica® brand laminate (The DillerCorporation, Cincinnati, Ohio). In some embodiments, the substrateincludes shower curtains or liners, upholstery, laundry, and carpeting.In some embodiments, the substrate includes a ship hull or a portionthereof. In some embodiments, the substrate includes a food contactsurface.

Biofilm preventing, removing or inhibiting coating compositions areprovided, including: (a) a film-forming resin; (b) a solvent thatdisperses said resin; (c) an effective amount of the compounds orcompositions disclosed herein, wherein said effective amount prevents orinhibits the growth of a biofilm thereon; and (d) optionally, at leastone pigment. In some embodiments, the compound is covalently coupled tothe resin. In some embodiments, the resin includes a polymeric material.

Substrates coated with coating composition disclosed herein are alsoprovided. In some embodiments, the substrate includes a polymericmaterial. In some embodiments, the substrate includes a solid support.In some embodiments, the substrate includes a drainpipe, glaze ceramic,porcelain, glass, metal, wood, chrome, plastic, vinyl, and Formica®brand laminate. In some embodiments, the substrate includes showercurtains or liners, upholstery, laundry, and carpeting. In someembodiments, the substrate includes a ship hull or a portion thereof. Insome embodiments, the substrate includes a food contact surface.

Methods of controlling biofilm formation on a substrate are provided,including the step of contacting the substrate with a compound and/orcomposition disclosed herein in an amount effective to inhibit biofilmformation. In some embodiments, controlling biofilm formation includesclearing a preformed biofilm from said substrate by administering aneffective amount of the compound and/or composition disclosed herein tosaid substrate, wherein said effective amount will reduce the amount ofsaid biofilm on said substrate. In some embodiments, the substrate mayinclude a drainpipe, glaze ceramic, porcelain, glass, metal, wood,chrome, plastic, vinyl, and Formica® brand laminate. In someembodiments, the substrate may include a food product (e.g., seafood).In some embodiments, the biofilm includes Gram-negative bacteria.

Methods for treating and/or preventing a bacterial infection (e.g.,chronic bacterial infection) in a subject in need thereof are provided,including administering to said subject a compound and/or compositiondisclosed herein in an amount effective to inhibit, reduce, or remove abiofilm component of said bacterial infection. The bacterial infectionmay include urinary tract infection, gastritis, respiratory infection,cystitis, pyelonephritis, osteomyelitis, bacteremia, skin infection,rosacea, acne, chronic wound infection, infectious kidney stones,bacterial endocarditis, and sinus infection.

Also provided are medical devices, including (a) a medical devicesubstrate; and (b) an effective amount of a compound disclosed herein,either coating the substrate, or incorporated into the substrate,wherein said effective amount prevents or inhibits the growth of abiofilm thereon. In some embodiments, the medical device substrate mayinclude stents, fasteners, ports, catheters, scaffolds and grafts. Insome embodiments, the compound is covalently coupled to said substrate.

Compounds and/or compositions for use in a method to control a biofilmare further provided. Also provided is the use of compounds and/orcompositions disclosed herein for the preparation of a medicament forthe treatment and/or prevention of a bacterial infection (e.g., chronicbacterial infection).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Biofilm development analysis by crystal violet staining (4-hbiofilm attachment assay). WT=no compound; 100=100 μg/mL; WT=nocompound; 200=200 μg/mL; WT=no compound; 300=300 μg/mL.

FIG. 2. Inhibition of biofilm formation by compounds 1 and 11.

FIG. 3. Planktonic growth studies. Diamonds are growth in the absence ofcompound, Triangles is bacteria grown in the presence of (1), Squaresdepict bacterial growth in the presence of (11).

FIG. 4. Bromoageliferin, TAGE and the TAGE derivative BromoTAGE.

FIG. 5. Inhibition of PDO300 biofilms with TAGE. Left, dose-response of[% Biofilm Formation] vs. TAGE concentration. Right, colony counts ofPDO300 grown in the absence and presence of TAGE (88 μM).

FIG. 6. Representative dose-response for PAO1 dispersion with TAGE.

FIG. 7. Dose-response comparison for BromoTAGE and DibromoTAGE againstA. baumannii.

FIG. 8. Dose-response comparison for BromoTAGE and DibromoTAGE againstRB50.

FIG. 9. Dose-response comparison for BromoTAGE and DibromoTAGE againstPAO1.

FIG. 10. Retrosynthetic analysis of oroidin and the RA scaffold.

FIG. 11. Members of the reverse amide library.

FIG. 12. Inhibition data at 500 μM for the RA library. All values areaverages of at least three experiments.

FIG. 13. Structure activity relationship of aliphatic chain RAanalogues.

FIG. 14. Dispersion of established P. aeruginosa biofilms with 12.

FIG. 15. Fragmentation of the oroidin template for SAR study.

FIG. 16. Region C SAR design.

FIG. 17. IC₅₀ values for the natural products oroidin 5 and sventrin 24.

FIG. 18. 2-amino-4-oxoimidazole analogues.

FIG. 19. Direct comparison of an atom-deletion effect in Region C.

FIG. 20. Natural product derivatives as anti-biofilm molecules,including dihydrosventrin (DHS).

FIG. 21. Dispersion of Proteobacterial Biofilms with DHS (7).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is further described below. All patent referencesreferred to in this patent application are hereby incorporated byreference in their entirety as if set forth fully herein.

A. Definitions

“Imidazole” refers to the commonly known stricture:

“H” refers to a hydrogen atom. “C” refers to a carbon atom. “N” refersto a nitrogen atom. “O” refers to an oxygen atom. “Halo” refers to F,Cl, Br or I. The term “hydroxy,” as used herein, refers to an —OHmoiety. “Br” refers to a bromine atom. “Cl” refers to a chlorine atom.“I” refers to an iodine atom. “F” refers to a fluorine atom.

An “acyl group” is intended to mean a —C(O)—R radical, where R is asuitable substituent (for example, an acetyl group, a propionyl group, abutyroyl group, a benzoyl group, or an alkylbenzoyl group).

“Alkyl,” as used herein, refers to a straight or branched chainhydrocarbon containing from 1 or 2 to 10 or 20 or more carbon atoms(e.g., C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15,etc.). Representative examples of alkyl include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2, 3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,n-decyl, and the like. In some embodiments, alkyl groups as describedherein are optionally substituted (e.g., from 1 to 3 or 4 times) withindependently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol,sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acidsidechain, amino acid and peptide.

The term “optionally substituted” indicates that the specified group iseither unsubstituted, or substituted by one or more suitablesubstituents. A “substituent” is an atom or atoms substituted in placeof a hydrogen atom on the parent chain or cycle of an organic molecule,for example, H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone,sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain,amino acid and peptide.

“Alkenyl,” as used herein, refers to a straight or branched chainhydrocarbon containing from 1 or 2 to 10 or 20 or more carbons, andcontaining at least one carbon-carbon double bond, formed structurally,for example, by the replacement of two hydrogens. Representativeexamples of “alkenyl” include, but are not limited to, ethenyl,2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl,2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl and the like. In someembodiments, alkenyl groups as described herein are optionallysubstituted (e.g., from 1 to 3 or 4 times) with independently selectedH, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone,sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain,amino acid and peptide.

“Alkynyl,” as used herein, refers to a straight or branched chainhydrocarbon group containing from 1 or 2 to 10 or 20 or more carbonatoms, and containing at least one carbon-carbon triple bond.Representative examples of alkynyl include, but are not limited, toacetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 1-butynyl andthe like. In some embodiments, alkynyl groups as described herein areoptionally substituted (e.g., from 1 to 3 or 4 times) with independentlyselected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone,sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain,amino acid and peptide.

The term “cycloalkyl,” as used herein, refers to a saturated cyclichydrocarbon group containing from 3 to 8 carbons or more. Representativeexamples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, cycloalkylgroups as described herein are optionally substituted (e.g., from 1 to 3or 4 times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

“Heterocyclo,” as used herein, refers to a monocyclic or a bicyclic ringsystem. Monocyclic heterocycle ring systems are exemplified by any 5 or6 member ring containing 1, 2, 3, or 4 heteroatoms independentlyselected from the group consisting of O, N, and S. The 5 member ring hasfrom 0 to 2 double bonds, and the 6 member ring has from 0-3 doublebonds. Representative examples of monocyclic ring systems include, butare not limited to, azetidine, azepine, aziridine, diazepine,1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline,imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole,isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline,oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine,pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine,pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine,tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole,thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine,thiophene, thiomorpholine, thiomorpholine sulfone, sulfoxide, thiopyran,triazine, triazole, trithiane, and the like. Bicyclic ring systems areexemplified by any of the above monocyclic ring systems fused to an arylgroup as defined herein, a cycloalkyl group as defined herein, oranother monocyclic ring system as defined herein. Representativeexamples of bicyclic ring systems include but are not limited to, forexample, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene,benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran,benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline,indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole,isoindoline, isoquinoline, phthalazine, pyranopyridine, quinoline,quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline,tetrahydroquinoline, thiopyranopyridine, and the like.

“Aryl” as used herein refers to a fused ring system having one or morearomatic rings. Representative examples of aryl include, azulenyl,indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.The aryl groups of this invention can be substituted with 1, 2, 3, 4, or5 substituents independently selected from alkenyl, alkenyloxy, alkoxy,alkoxyalkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy,alkylsulfinyl, alkylsulfonyl, alkylthio, alkynyl, aryl, aryloxy, azido,arylalkoxy, arylalkyl, aryloxy, carboxy, cyano, formyl, halogen,haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, mercapto, nitro, sulfamyl,sulfo, sulfonate, —NR′R″ (wherein, R′ and R″ are independently selectedfrom hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl and formyl), and—C(O)NR′R″ (wherein R′ and R″ are independently selected from hydrogen,alkyl, alkylcarbonyl, aryl, arylalkyl, and formyl).

“Heteroaryl” means a cyclic, aromatic hydrocarbon in which one or morecarbon atoms have been replaced with heteroatoms. If the heteroarylgroup contains more than one heteroatom, the heteroatoms may be the sameor different. Examples of heteroaryl groups include pyridyl,pyrimidinyl, imidazolyl, thienyl, furyl, pyrazinyl, pyrrolyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, indolyl, isoindolyl, indolizinyl,triazolyl, pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, isothiazolyl, andbenzo[b]thienyl. Preferred heteroaryl groups are five and six memberedrings and contain from one to three heteroatoms independently selectedfrom the group consisting of: O, N, and S. The heteroaryl group,including each heteroatom, can be unsubstituted or substituted with from1 to 4 suitable substituents, as chemically feasible. For example, theheteroatom S may be substituted with one or two oxo groups, which may beshown as ═O.

“Alkoxy,” as used herein, refers to an alkyl group, as defined herein,appended to the parent molecular moiety through an oxy group, as definedherein. Representative examples of alkoxy include, but are not limitedto, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy,hexyloxy and the like. In some embodiments, alkoxy groups as describedherein are optionally substituted (e.g., from 1 to 3 or 4 times) withindependently selected H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol,sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acidsidechain, amino acid and peptide.

An “amine” or “amino” group is intended to mean the radical —NH₂.“Optionally substituted” amines refers to —NH₂ groups wherein none, oneor two of the hydrogens is replaced by a suitable substituent asdescribed herein, such as alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, carbonyl, carboxy, etc. In someembodiments, one or two of the hydrogens are optionally substituted withindependently selected, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol,sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acidsidechain, amino acid and peptide. Disubstituted amines may havesubstituents that are bridging, i.e., form a heterocyclic ring structurethat includes the amine nitrogen.

An “amide” as used herein refers to an organic functional group having acarbonyl group (C∇O) linked to a nitrogen atom (N), or a compound thatcontains this group, generally depicted as:

wherein, R and R′ can independently be any covalently-linked atom oratoms, for example, H, halo, hydroxy, acyl, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol,sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acidsidechain, amino acid and peptide.

A “thiol” or “mercapto” refers to an —SH group or to its tautomer ═S.

A “sulfone” as used herein refers to a sulfonyl functional group,generally depicted as:

wherein, R can be any covalently-linked atom or atoms, for example, H,halo, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo,aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo,oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide.

A “sulfoxide” as used herein refers to a sulfinyl functional group,generally depicted as:

wherein, R can be any covalently-linked atom or atoms, for example, H,halohydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo,aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo,oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide.

The term “oxo,” as used herein, refers to a ═O moiety. The term “oxy,”as used herein, refers to a —O— moiety.

“Nitro” refers to the organic compound functional group —NO₂.

“Carbonyl” is a functional group having a carbon atom double-bonded toan oxygen atom (—C═O). “Carboxy” as used herein refers to a —COOHfunctional group, also written as —(C═O)—OH.

“Amino acid sidechain” as used herein refers to any of the 20 commonlyknown groups associated with naturally-occurring amino acids, or anynatural or synthetic homologue thereof. An “amino acid” includes thesidechain group and the amino group, alpha-carbon atom, and carboxygroups, as commonly described in the art. Examples of amino acidsinclude glycine, and glycine that is substituted with a suitablesubstituent as described herein, such as alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, carbonyl, carboxy,etc., or a pharmaceutically acceptable salt or prodrug thereof. Forexample, “Histidine” is one of the 20 most commonly known amino acidsfound naturally in proteins. It contains an imidazole side chainsubstituent. Other examples of naturally-occurring amino acids includelysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine,serine, threonine, tyrosine, alanine, valine, leucine, isoleucine,phenylalanine, methionine, cryptophan, and cysteine. Also included inthe definitions of “amino acid sidechain” and “amino acid” is proline,which is commonly included in the definition of an amino acid, but istechnically an imino acid. As used in this application, both thenaturally-occurring L-, and the non-natural D-amino acid enantiomers areincluded. A “peptide” is a linear chain of amino acids covalently linkedtogether, typically through an amide linkage, and contains from 1 or 2to 10 or 20 or more amino acids, and is also optionally substitutedand/or branched.

A “pharmaceutically acceptable salt” is intended to mean a salt thatretains the biological effectiveness of the free acids and bases of aspecified compound and that is not biologically or otherwiseundesirable. Examples of pharmaceutically acceptable salts includesulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogenphosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates,citrates, lactates, γ-hydroxybutyrates, glycollates, tartrates,methane-sulfonates, propanesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, and mandelates.

A “prodrug” is intended to mean a compound that is converted underphysiological conditions or by solvolysis or metabolically to aspecified compound that is pharmaceutically active. A thoroughdiscussion is provided in T. Higuchi and V. Stella, Prodrugs as Noveldelivery Systems, Vol. 14 of the A.C.S. Symposium Series and in EdwardB. Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated by reference herein in their entirety.

B. Active Compounds

In some of the embodiments provided in the present invention, activecompounds are provided. These active compounds are derivatives ofimidazole. Active compounds as described herein can be prepared asdetailed below or in accordance with known procedures or variationsthereof that will be apparent to those skilled in the art.

As will be appreciated by those of skill in the art, the activecompounds of the various formulas disclosed herein may contain chiralcenters, e.g. asymmetric carbon atoms. Thus, the present invention isconcerned with the synthesis of both: (i) racemic mixtures of the activecompounds, and (ii) enantiomeric forms of the active compounds. Theresolution of racemates into enantiomeric forms can be done inaccordance with known procedures in the art. For example, the racematemay be converted with an optically active reagent into a diastereomericpair, and the diastereomeric pair subsequently separated into theenantiomeric forms.

Geometric isomers of double bonds and the like may also be present inthe compounds disclosed herein, and all such stable isomers are includedwithin the present invention unless otherwise specified. Also includedin active compounds of the invention are tautomers (e.g., tautomers ofimidazole) and rotamers.

Active compounds for carrying out the present invention includecompounds of Formula (I):

wherein:

R¹ and R² and R³ are each independently selected from the groupconsisting of: H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone,sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain,amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

In some embodiments of Formula (I), R¹ is an amino and R² is H, depictedas Formula (I)(a):

wherein:

R³ is selected from the group consisting of: H, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy,amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

In some embodiments of Formula (I)(a), R³ comprises amino acidsidechains. Examples of these embodiments are depicted in Formulas(I)(a)(i)-(I)(a)(ix):

In some embodiments of Formula (I)(a), R³ comprises amino alkanes oramino alkenes. Examples of these embodiments are depicted in(I)(a)(xi)-(I)(a)(xiv):

In some embodiments of Formula (I)(a), R³ comprises an alkyl or alkenylwith disubstituted amides. Examples of these embodiments are depicted inFormulas (I)(a) (xv)-(I)(a)(xviii):

wherein:

R⁴ R⁵, R⁶, and R⁷ are each independently selected from the groupconsisting of: H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, sulfone, sulfoxide,oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

In some embodiments of Formulas (I)(a)(xv)-(I)(a)(xviii), R⁴, R⁵, R⁶,and R⁷ comprise aryls or heteroaryls. Examples of these embodimentsinclude those aryls and heteroaryls depicted in Formulas(II)(b)(i)-(II)(b)(ix) below for constituents R¹¹ and R¹².

In some embodiments of Formula (I)(a), R³ comprises alkyls withheterocycloalkyls, optionally substituted with further alkyls oralkenyls. Examples of these embodiments are depicted in Formulas(I)(a)(xix)-(I)(a)(xx):

Active compounds for carrying out the present invention includecompounds of Formula (II):

wherein:

R⁸ is selected from the group consisting of: H, amino, hydroxy, andthiol; and

R⁹ and R¹⁰ are each independently selected from the group consisting of:H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo,aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo,oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

Some embodiments of the active compounds comprise derivatives of2-aminoimidazole. For example, in some embodiments of Formula (II), R⁸comprises an amino, R⁹ and R¹⁰ are the same, and R⁹ and R¹⁰ comprise H.Examples of these embodiments are depicted in Formula (II)(a):

Examples of certain stereoisomers of Formula (II)(a) include thosedepicted in Formulas (II)(a)(i)-(II)(a)(ii):

The discussion herein is, for simplicity, given without furtherreference to stereoisomerism. However, as noted above, the activecompounds of the various formulas disclosed herein contain chiralcenters, e.g. asymmetric carbon atoms. Thus, the present invention isconcerned with the synthesis of both: (i) racemic mixtures of the activecompounds, and (ii) enantiomeric forms of the active compounds. Theresolution of racemates into enantiomeric forms can be done inaccordance with known procedures in the art. For example, the racematemay be converted with an optically active reagent into a diastereomericpair, and the diastereomeric pair subsequently separated into theenantiomeric forms.

In some embodiments of Formula (II), R⁸ comprises an amino, and R⁹ andR¹⁰ comprise carbonyls, generally depicted in Formula (II)(b):

wherein:

R¹¹ and R¹² are each independently selected from the group consistingof: H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo,aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo,oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

In some embodiments of Formula (II)(b), R¹¹ and R¹² are the same, andR¹¹ and R¹² comprise aryls or heteroaryls Examples of these embodimentsare depicted in Formulas (II)(b)(i)-(II)(b)(ix):

In some embodiments of Formula (II), R⁸ comprises an amino, R⁹ and R¹⁰are the same, and R⁹ and R¹⁰ comprise sulfones, generally depicted inFormula (II)(c):

wherein:

R¹³ and R¹⁴ are each independently selected from the group consistingof: H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo,aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo,oxy, nitro, carbonyl, carboxy, amino acid sidechain, amino acid andpeptide;

or a pharmaceutically acceptable salt or prodrug thereof Each group canbe optionally substituted.

In some embodiments of Formula (II)(c), R¹³ and R¹⁴ are the same, andR¹³ and R¹⁴ comprise aryls or heteroaryls. An example of theseembodiments is depicted in Formula (II)(c)(i):

Active compounds for carrying out the present invention includecompounds of Formula (III):

wherein:

R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²² are each independentlyselected from the group consisting of: H, hydroxy, acyl, alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy, amino,amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl, carboxy,amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

Active compounds for carrying out the present invention includecompounds of Formula (IV):

wherein:

R²³ is selected from the group consisting of: H, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

In some embodiments of Formula (IV), R²³ comprises an amino acidsidechain. Examples of these embodiments are depicted in Formula (IV)(a)through Formula (IV)(c). The amino acids or peptides are optionallysubstituted, exemplified in Formula (IV)(d):

Active compound embodiments include those depicted by Formula (V) andFormula (VI):

These formulas are also optionally substituted.

Active compounds also include those represented by Formula (X):

wherein:

R¹ and R² and R³ are each independently selected from the groupconsisting of: H, hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclo, aryl, heteroaryl, alkoxy, amino, amide, thiol, sulfone,sulfoxide, oxo, oxy, nitro, carbonyl, carboxy, amino acid sidechain,amino acid and peptide; and

A and B are each independently selected from N, S and O.

or a pharmaceutically acceptable salt or prodrug thereof. Each group canbe optionally substituted.

In some embodiments of Formula (X), R1 is amino; R3 is H; and A and Bare each N, generally depicted by Formula (X)(I)(a):

wherein R⁵ is an alkyl, alkenyl or alkynyl having an amide groupsubstituted thereon;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(a) are represented by Formula(X)(I)(a)(1):

wherein:

n is 1 to 10 carbons, saturated or unsaturated; and

R⁶ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl. heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

A preferred embodiment of Formula (X)(I)(a) is represented by Formula(X)(I)(a)(1)(A):

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(a) are represented by Formula(X)(I)(a)(2):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and

R⁷ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(a)(2) are represented by Formula(X)(I)(a)(2)(A):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and X, Y and Z are each independently selected H, halo,hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(a)(2)(A) are represented by Formula(X)(I)(a)(2)(A)(i):

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

In some embodiments of Formula (X), R1 is amino; R3 is H; and A is S andB is N, generally depicted by Formula (X)(I)(b):

wherein R⁸ is an alkyl, alkenyl or alkynyl having an amide groupsubstituted thereon;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(b) are represented by Formula(X)(I)(b)(1):

wherein:

n is 1 to 10 carbons, saturated or unsaturated; and

R⁹ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

A preferred embodiment of Formula (X)(I)(b) is represented by Formula(X)(I)(b)(1)(A):

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(b) are represented by Formula(X)(I)(b)(2):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and

R¹⁰ is selected form the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(b)(2) are represented by Formula(X)(I)(b)(2)(A):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and X, Y and Z are each independently selected H, halo,hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(b)(2)(A) are represented by Formula(X)(I)(b)(2)(A)(i):

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

In some embodiments of Formula (X), R1 is thiol; R3 is H; and A and Bare each N, generally depicted by Formula (X)(I)(c):

wherein R¹¹ is an alkyl, alkenyl or alkynyl having an amide groupsubstituted thereon;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(c) are represented by Formula(X)(I)(c)(1):

wherein:

n is 1 to 10 carbons, saturated or unsaturated; and

R¹² is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodding thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

A preferred embodiment of Formula (X)(I)(c) is represented by Formula(X)(I)(c)(1)(A):

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(a) are represented by Formula(X)(I)(a)(2):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and

R¹³ is selected from the group consisting of H, halo, hydroxy, acyl,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl,alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro,carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(c)(2) are represented by Formula(X)(I)(c)(2)(A):

wherein:

n is 1 to 10 carbons, saturated or unsaturated, substituted orunsubstituted; and X, Y and Z are each independently selected H, halo,hydroxy, acyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,heteroaryl, alkoxy, amino, amide, thiol, sulfone, sulfoxide, oxo, oxy,nitro, carbonyl, carboxy, amino acid sidechain, amino acid and peptide;

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

Some embodiments of Formula (X)(I)(c)(2)(A) are represented by Formula(X)(I)(c)(2)(A)(i):

or a pharmaceutically acceptable salt or prodrug thereof.

This formula may be optionally substituted (e.g., from 1 to 3 or 4times) with independently selected H, halo, hydroxy, acyl, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl, heteroaryl, alkoxy,amino, amide, thiol, sulfone, sulfoxide, oxo, oxy, nitro, carbonyl,carboxy, amino acid sidechain, amino acid and peptide.

C. Compositions

In some embodiments, biofilm preventing, removing or inhibitingcompositions are provided, comprising a carrier and an effective amountof active compound. “Biofilm” or “biofilms” refer to communities ofmicroorganisms that are attached to a substrate. The microorganismsoften excrete a protective and adhesive matrix of polymeric compounds.They often have structural heterogeneity, genetic diversity, and complexcommunity interactions. “Biofilm preventing”, “biofilm removing”,“biofilm inhibiting”, “biofilm reducing”, “biofilm resistant”, “biofilmcontrolling” or “antifouling” refer to prevention of biofilm formation,inhibition of the establishment or growth of a biofilm, or decrease inthe amount of organisms that attach and/or grow upon a substrate, tip toand including the complete removal of the biofilm. As used herein, a“substrate” can include any living or nonliving structure. For example,biofilms often grow on synthetic materials submerged in an aqueoussolution or exposed to humid air, but they also can form as floatingmats on a liquid surface, in which case the microorganisms are adheringto each other or to the adhesive matrix characteristic of a biofilm. An“effective amount” of a biofilm preventing, removing or inhibitingcomposition is that amount which is necessary to carry out thecomposition's function of preventing, removing or inhibiting a biofilm.

In some embodiments, the carrier is a pharmaceutically acceptablecarrier. A “pharmaceutically acceptable carrier” as used herein refersto a composition that, when combined with an active compound of thepresent invention, facilitates the application or administration of thatactive compound for its intended purpose to prevent or inhibit biofilmformation, or remove an existing biofilm. The active compounds describedabove may be formulated for administration in a pharmaceuticallyacceptable carrier in accordance with known techniques. See, e.g.,Remington, The Science And Practice of Pharmacy (9^(th) Ed. 1995). Thepharmaceutically acceptable carrier must, of course, also be acceptablein the sense of being compatible with any other ingredients in thecomposition. The carrier may be a solid or a liquid, or both, and ispreferably formulated with the compound as a unit-dose composition, forexample, a tablet, which may contain from 0.01 or 0.5% to 95% or 99% byweight of the active compound. One or more active compounds may beincorporated in the compositions of the invention, which may be preparedby any of the well-known techniques of pharmacy comprising admixing thecomponents, optionally including one or more accessory ingredients.

In general, compositions of the invention are prepared by uniformly andintimately admixing the active compound with a liquid or finely dividedsolid carrier, or both, and then, if necessary, shaping the resultingmixture For example, a tablet may be prepared by compressing or moldinga powder or granules containing the active compound, optionally with oneor more accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the compound in a free-flowing form,such as a powder or granules optionally mixed with a binder, lubricant,inert diluent, and/or surface active/dispersing agent(s). Molded tabletsmay be made by molding, in a suitable machine, the powdered compoundmoistened with an inert liquid binder.

The compositions of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used. Preferred routes of parenteral administration includeintrathecal injection and intraventricular injection into a ventricle ofthe brain.

Compositions suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchcompositions may be prepared by any suitable method of pharmacy, whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above).

Compositions suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavored base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Compositions of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes thatrender the composition isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The compositions may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.

For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound ofFormula (I), or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate that is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent that is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Compositions suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by mixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Compositions suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers that may be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Compositions suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Compositionssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable compositions comprise citrateor bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2Mof the active ingredient.

Also provided in some embodiments are biofilm preventing, removing orinhibiting compositions comprising an active compound and an additionalbiocide that is not an active compound in the group herein disclosed ofimidazole derivatives. “Biocide” as used herein refers to a substancewith the ability to kill or to inhibit the growth of microorganisms.Common biocides include oxidizing and non-oxidizing chemicals. Examplesof oxidizing biocides include chlorine, chlorine dioxide, and ozone.Examples of non-oxidizing biocides include quaternary ammoniumcompounds, formaldehyde, and anionic and non-anionic surface agents.Chlorine is the most common biocide used in sanitizing water systems.Antibiotics can also be biocides. Common antibiotics includeaminoglycosides, cephalosporins, glycopeptides, macrolides, penicillins,polypeptides, sulfonamides, tetracyclines, etc. Antibiotics treatinfections by either killing or preventing the growth of bacteria.

In some embodiments, a dentifrice composition is provided comprising theactive compounds. A “dentifrice” is a substance that is used to cleanthe teeth. It may be in the form of, e.g., a paste or powder. Commonlyknown dentifrices include toothpaste, mouthwash, chewing gum, dentalfloss, and dental cream. Other examples of dentifrices includetoothpowder, mouth detergent, troches, dental or gingival massage cream,dental strips, dental gels, and gargle tablets. Examples of dentifricecompositions comprising toothpaste and mouthwash are found in U.S. Pat.No. 6,861,048 (Yu et al.); U.S. Pat. No. 6,231,836 (Takhtalian et al.);and U.S. Pat. No. 6,331,291 (Glace et al.); each incorporated byreference herein in their entirety.

A coating composition is also provided. A “coating” as used herein isgenerally known. Any of a variety of organic and aqueous coatingcompositions, with or without pigments, may be modified to containbiofilm inhibiting compositions as described herein, including but notlimited to those described in U.S. Pat. Nos. 7,109,262, 6,964,989,6,835,459, 6,677,035, 6,528,580, 6,235,812, etc., each incorporated byreference herein in their entirety.

In general, the coatings comprise a film-forming resin, an aqueous ororganic solvent that disperses the resin; and, optionally, at least onepigment. Other ingredients such as colorants, secondary pigments,stabilizers and the like can be included if desired. However, for use inthe present invention the compositions further comprise one or morebiofilm inhibiting compounds as described herein, which may be carriedby or dispersed in the solvent and/or resin, so that the biofilminhibiting compounds are dispersed or distributed on the substrate anarticle coated. A resin may carry the biofilm inhibiting compoundsthrough covalent attachment through means well known in the art. Theresin may comprise, for example, a polymeric material. A polymericmaterial is a material that is comprised of large molecules made fromassociated smaller repeating structural units, often covalently linked.Common examples of polymeric materials are unsaturated polyester resins,and epoxy resins.

Any suitable article can be coated, in whole or in part, with acomposition of the invention. Suitable articles include, but are notlimited to, automobiles and airplanes (including substrates such as wingand propeller surfaces for aerodynamic testing), vessel hulls (includinginterior and exterior surfaces thereof), pressure vessels (includinginterior and exterior surfaces thereof) medical implants, windmills,etc. Coating of the article with the composition can be carried out byany suitable means, such as by brushing, spraying, electrostaticdeposition, dip coating, doctor blading, etc.

D. Methods of Use

Methods of controlling biofilm formation on a substrate are disclosed,comprising the step of administering an active compound to a substratein an amount effective to inhibit biofilm formation. A “substrate” asused herein is a base on which an organism, such as those commonly foundin biofilms, may live. The term “substrate,” as used herein, refers toany substrate, whether in an industrial or a medical setting, thatprovides or can provide an interface between an object and a fluid,permitting at least intermittent contact between the object and thefluid. A substrate, as understood herein, further provides a plane whosemechanical structure, without further treatment, is compatible with theadherence of microorganisms. Substrates compatible with biofilmformation may be natural or synthetic, and may be smooth or irregular.Fluids contacting the substrates can be stagnant or flowing, and canflow intermittently or continuously, with laminar or turbulent or mixedflows. A substrate upon which a biofilm forms can be dry at times withsporadic fluid contact, or can have any degree of fluid exposureincluding total immersion. Fluid contact with the substrate can takeplace via aerosols or other means for air-borne fluid transmission.

Biofilm formation with health implications can involve those substratesin all health-related environments, including substrates found inmedical environments and those substrates in industrial or residentialenvironments that are involved in those functions essential to humanwell being, for example, nutrition, sanitation and the prevention ofdisease. Substrates found in medical environments include the inner andouter aspects of various instruments and devices, whether disposable orintended for repeated uses. Examples include the entire spectrum ofarticles adapted for medical use, including scalpels, needles, scissorsand other devices used in invasive surgical, therapeutic or diagnosticprocedures; implantable medical devices, including artificial bloodvessels, catheters and other devices for the removal or delivery offluids to patients, artificial hearts, artificial kidneys, orthopedicpins, plates and implants; catheters and other tubes (includingurological and biliary tubes, endotracheal tubes, peripherablyinsertable central venous catheters, dialysis catheters, long termtunneled central venous catheters, peripheral venous catheters, shortterm central venous catheters, arterial catheters, pulmonary catheters,Swan-Ganz catheters, urinary catheters, peritoneal catheters), urinarydevices (including long term urinary devices, tissue bonding urinarydevices, artificial urinary sphincters, urinary dilators), shunts(including ventricular or arterio-venous shunts); prostheses (includingbreast implants, penile prostheses, vascular grafting prostheses, heartvalves, artificial joints, artificial larynxes, otological implants),vascular catheter ports, wound drain tubes, hydrocephalus shunts,pacemakers and implantable defibrillators, and the like. Other exampleswill be readily apparent to practitioners in these arts. Substratesfound in the medical environment also include the inner and outeraspects of pieces of medical equipment, medical gear worn or carried bypersonnel in the health care setting. Such substrates can includecounter tops and fixtures in areas used for medical procedures or forpreparing medical apparatus, tubes and canisters used in respiratorytreatments, including the administration of oxygen, of solubilized drugsin nebulizers and of anesthetic agents. Also included are thosesubstrates intended as biological barriers to infectious organisms inmedical settings, such as gloves, aprons and faceshields. Commonly usedmaterials for biological barriers may be latex-based or non-latex based.Vinyl is commonly used as a material for non-latex surgical gloves.Other such substrates can include handles and cables for medical ordental equipment not intended to be sterile. Additionally, suchsubstrates can include those non-sterile external substrates of tubesand other apparatus found in areas where blood or body fluids or otherhazardous biomaterials are commonly encountered.

Substrates in contact with liquids are particularly prone to biofilmformation. As an example, those reservoirs and tubes used for deliveringhumidified oxygen to patients can bear biofilms inhabited by infectiousagents. Dental unit waterlines similarly can bear biofilms on theirsubstrates, providing a reservoir for continuing contamination of thesystem of flowing an aerosolized water used in dentistry. Sprays,aerosols and nebulizers are highly effective in disseminating biofilmfragments to a potential host or to another environmental site. It isespecially important to health to prevent biofilm formation on thosesubstrates from where biofilm fragments can be carried away by sprays,aerosols or nebulizers contacting the substrate.

Other substrates related to health include the inner and outer aspectsof those articles involved in water purification, water storage andwater delivery, and articles involved in food processing. Substratesrelated to health can also include the inner and outer aspects of thosehousehold articles involved in providing for nutrition, sanitation ordisease prevention. Examples can include food processing equipment forhome use, materials for infant care, tampons and toilet bowls.“Substrate” as used herein also refers to a living substrate, such asthe inner ear of a patent.

Substrates can be smooth or porous, soft or hard. Substrates can includea drainpipe, glaze ceramic, porcelain, glass, metal, wood, chrome,plastic, vinyl, Formica® brand laminate, or any other material that mayregularly come in contact with an aqueous solution in which biofilms mayform and grow. The substrate can be a substrate commonly found onhousehold items such as shower curtains or liners, upholstery, laundry,and carpeting.

A substrate on which biofilm preventing, removing or inhibiting isimportant is that of a ship hull. Biofilms, such as those of Halomonaspacifica, promote the corrosion of the hull of ships and also increasethe roughness of the hull, increasing the drag on the ship and therebyincreasing fuel costs. The biofilm can also promote the attachment oflarger living structures such as barnacles on the ship hull. Fuel canaccount for half of the cost of marine shipping, and the loss in fuelefficiency due to biofilm formation is substantial.

Substrates on which biofilms can adhere include those of livingorganisms, as in the case of humans with chronic infections caused bybiofilms, as discussed above. Biofilms can also form on the substratesof food contact surfaces, such as those used for processing seafood, andalso on food products themselves. Examples of seafood products that mayhave biofilm contamination include oysters. Human infections caused bythe ingestion of raw oysters has been linked to Vibrio vulnificusbacterium. Vibrio bacteria attach to algae and plankton in the water andtransfer to the oysters and fish that feed on these organisms.

Other examples of substrates or devices on which biofilms can adhere canbe found in U.S. Pat. Nos. 5,814,668 and 7,087,661; and U.S. Pat.Application Publication Nos. 2006/0228384 and 2006/0018945, each ofwhich is incorporated herein by reference in their entirety.

Also disclosed is a method of controlling biofilm formation wherein thebiofilm comprises Gram-negative bacteria. “Gram-negative” bacteria arethose that do not retain crystal violet dye after an alcohol wash in theGram staining protocol. This is due to structural properties in the cellwalls of the bacteria. Many genera and species of Gram-negative bacteriaare pathogenic. Gram-negative bacteria include members of the phylumproteobacteria, which include genus members Escherichia, Salmonella,Vibrio, and Helicobacter. A “genus” is a category of biologicalclassification ranking between the family and the species, comprisingstructurally or phylogenetically related species, or an isolated speciesexhibiting unusual differentiation. It is usually designated by a Latinor latinized capitalized singular noun. Examples of genera ofbiofilm-forming bacteria affected by active compounds of this inventioninclude, but are not limited to, Pseudomonas, Bordetella, Vibrio,Haemophilus, Halomonas, and Acinetobacter. “Species” refer to a categoryof biological classification ranking below the genus, and comprisemembers that are structurally or phylogenetically related, or anisolated member exhibiting unusual differentiation. Species are commonlydesignated by a two-part name, which name includes the capitalized anditalicized name of the genus in which the species belongs as the firstword in the name, followed by the second word that more specificallyidentifies the member of the genus, which is not capitalized. Examplesof species of bacteria capable of forming biofilms that are affected byactive compounds of the present invention include Pseudomonasaeuroginosa, Bordetella pertussis, Vibrio vulnificus, Haemophilusinfluenzae, Halomonas pacifica, and Acinetobacter baumannii.

Other examples of Gram-negative bacteria affected by active compounds ofthe present invention include, but are not limited to, bacteria of thegenera Klebsiella, Proteus, Neisseria, Helicobacter, Brucella,Legionella, Campylobacter, Francisella, Pasteurella, Yersinia,Bartonella, Bacteroides, Streptobacillus, Spirillum, Moraxella andShigella. Examples of Gram-positive bacteria affected by activecompounds of the present invention include, but are not limited to,bacteria of the genera Listeria, Staphylococcus, Streptococcus,Bacillus, Corynebacterium, Peptostreptococcus, and Clostridium.Furthermore, bacteria affected by active compounds of the presentinvention includes Gram-positive bacteria including, but not limited to,Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes,Streptococcus pneumoniae, Bacillus cereus, Bacillus anthracis,Clostridium botulinum, Clostridium perfringens, Clostridium difficile,Clostridium tetani, Corynebacterium diphtheriae, Corynebacteriumulcerans, and Peptostreptococcus anaerobius. Additional bacteriaaffected by active compounds of the present invention also includebacterial genera including, but not limited to, Actinomyces,Propionibacterium, Nocardia and Streptomyces.

A method for treating a chronic bacterial infection in a subject in needthereof is disclosed, comprising administering active compound to saidsubject in an amount effective to inhibit, reduce, or remove a biofilmcomponent of said chronic bacterial infection. “Treating” as used hereinrefers to any type of activity that imparts a benefit to a patientafflicted with a disease, including improvement in the condition of thepatient (e.g., in one or more symptoms), delay in the progression of thedisease, delay in onset of the disease, etc. The present invention isprimarily concerned with the treatment of human subjects, but theinvention may also be carried out on animal subjects, particularlymammalian subjects (e.g., mice, rats, dogs, cats, rabbits, and horses),avian subjects (e.g., parrots, geese, quail, pheasant), livestock (e.g.,pigs, sheep, goats, cows, chickens, turkey, duck, ostrich, emu), reptileand amphibian subjects, for veterinary purposes or animal husbandry, andfor drug screening and drug development purposes.

A “chronic bacterial infection” is a bacterial infection that is of along duration or frequent recurrence. For example, a chronic middle earinfection, or otitis media, can occur when the Eustachian tube becomesblocked repeatedly due to allergies, multiple infections, ear trauma, orswelling of the adenoids. The definition of “long duration” will dependupon the particular infection. For example, in the case of a chronicmiddle ear infection, it may last for weeks to months. Other knownchronic bacterial infections include urinary tract infection (mostcommonly caused by Escherichia coli and/or Staphylococcussaprophyticus), gastritis (most commonly caused by Helicobacter pylori),respiratory infection (such as those commonly afflicting patents withcystic fibrosis, most commonly caused by Pseudomonas aeuroginosa),cystitis (most commonly caused by Escherichia coli), pyelonephritis(most commonly caused by Proteus species, Escherichia coli and/orPseudomonas species), osteomyelitis (most commonly caused byStaphylococcus aureus, but also by Escherichia coli), bacteremia, skininfection, rosacea, acne, chronic wound infection, infectious kidneystones (can be caused by Proteus mirabilis), bacterial endocarditis, andsinus infection. A common infection afflicting pigs is atrophic rhinitis(caused by Bordatella species, e.g. Bordatella rhinitis).

Various nosocomial infections that are especially prevalent in intensivecare units implicate Acinetobacter species such as Acinetobacterbaumannii and Acinetobacter lwoffi. Acinetobacter baumanni is a frequentcause of nosocomial pneumonia, and can also cause skin and woundinfections and bacteremia. Acinetobacter lwoffi causes meningitis. TheAcinetobacter species are resistant to many classes of antibiotics. TheCDC has reported that bloodstream infections implicating Acinetobacterbaumanni were becoming more prevalent among service members injuredduring the military action in Iraq and Afghanistan.

Also disclosed is a method of clearing a preformed biofilm from asubstrate comprising the step of administering an effective amount ofcompound to said substrate, wherein said effective amount will reducethe amount of said biofilm on said substrate. “Preformed biofilm” is abiofilm that has begun to adhere to a substrate. The biofilm may or maynot yet be fully formed.

E. Devices

Medical devices comprising a Substrate and an effective amount of activecompounds are also disclosed. “Medical device” as used herein refers toan object that is inserted or implanted in a subject or applied to asurface of a subject. Common examples of medical devices include stents,fasteners, ports catheters, scaffolds and grafts. A “medical devicesubstrate” can be made of a variety of biocompatible materials,including, but not limited to, metals, ceramics, polymers, gels andfluids not normally found within the human body. Examples of polymersuseful in fabricating medical devices include such polymers assilicones, rubbers, latex, plastics polyanhydrides, polyesters,polyorthoesters, polyamides, polyacrylonitrile, polyurethanes,polyethylene, polytetrafluoroethylene, polyethylenetetraphthalate, etc.Medical devices can also be fabricated using certain naturally-occurringmaterials or treated with naturally-occurring materials. Medical devicescan include any combination of artificial materials, combinationsselected because of the particular characteristics of the components.Medical devices can be intended for short-term or long-term residencewhere they are positioned. A hip implant is intended for several decadesof use, for example. By contrast, a tissue expander may only be neededfor a few months, and is removed thereafter.

Some examples of medical devices are found in U.S. Pat. No. 7,081,133(Chinn et al.); U.S. Pat. No. 6,562,295 (Neuberger); and U.S. Pat. No.6,387,363 (Gruskin); each incorporated by reference herein in theirentirety.

F. Covalent Coupling of Active Compounds

Active compounds as described herein can be covalently coupled tosubstrates. Examples of substrates include solid supports and polymers.The polymers, typically organic polymers, may be in solid form, liquidform, dispersed or solubilized in a solvent (e.g., to form a coatingcomposition as described above), etc. The solid support may include thesubstrate examples as described above to be coated with or treated withactive compounds of the invention.

Covalent coupling can be carried out by any suitable technique. Activecompounds of the present invention may be appended to a substrate viaaldehyde condensation, amide or peptide bond, carbon-carbon bond, or anysuitable technique commonly used in the art.

For example, the active compound of Formula (II)(a) can be covalentlylinked to a substrate having a carboxylic acid via peptide coupling asshown (with the dark bar representing the substrate):

The active compound of Formula (I)(a)(i) can be covalently linked to asubstrate by condensation with an aldehyde as shown:

Various coupling reactions can be used to covalently link activecompounds of the present invention to a substrate. Examples of couplingreactions that can be used include, but are not limited to, Hiyama,Suzuki, Sonogashira, Heck, Stille, Negishi, Kumada, Wurtz, Ullmann,Cadiot-Chodkiewicz, Buchwald-Hartwig, and Grignard reactions. Forexample, an active compound of Formula (I)(a)(iv) that is substitutedwith a halide (e.g. bromo or chloro) can be coupled to a substrate via aHeck reaction as shown:

This listing of examples of reactions that can be used to append activecompounds of the present invention to a substrate is not intended to beexhaustive. Those skilled in the art will readily appreciate variousother methods of carrying out these teachings. Further examples andexplanations of these types of reactions can be found in U.S. Pat. No.6,136,157 (Lindeberg et al.) and U.S. Pat. No. 7,115,653 (Baxter etal.), which are each hereby incorporated by reference in their entirety.

Some aspects of the present invention are described in more detail inthe following non-limiting examples.

EXAMPLE 1

Inhibition of Pseudomonas aeruginosa biofilm formation. A compound ofFormula (II)(a)(i) (“compound 1”) was synthesized in 10 linear steps,outlined in Scheme 1. Diethyl fumarate and 1,3-butadiene were subjectedto a [4+2] cycloaddition to yield the Diels-Alder adduct 2. The diester2 was then reduced with lithium aluminum hydride (LiAlH₄) to yield diol3. The diol was then treated with mesityl chloride (MsCl) to generatethe corresponding bis-mesylate 4 that was then refluxed with sodiumazide (NaN₃) to yield di-azide 5. We then epoxidized 5 withmeta-chloroperoxybenzoic acid (m-CPBA) at room temperature in theabsence of ambient light to generate 6. Epoxide 6 was then treated withNaN₃ and sulfuric acid H₂SO₄ in refluxing ethanol to yield theazidoalcohol 7 that was subsequently subjected to hydrogenatingconditions in the presence of di-tert-butyl dicarbonate (Boc₂O). Thetri-Boc protected amino alcohol 8 was then oxidized with pyridiniumchlorochromate to generate ketone 9. Quantitative Boc-deprotection withTFA, followed by conversion to the HCl, and finally condensation of ourα-aminoketone with cyanamide generated 1 in 7.5% overall yield fromcommercially available starting materials.

Control compounds were also synthesized (Scheme 2). Starting with2-aminoimidazole 1, we acylated the 2-amino position of the2-aminoimidazole ring with an acyl pyrrole moiety to yield compound 10.This was designed to test the importance of the 2-AI ring as thecritical pharmacophore that imparted biological activity on ourmolecule. We also synthesized the diastereomer of compound 1, where oneof the chiral centers was inverted. This diastereomer, 11, wassynthesized using the same synthetic sequence that we delineated abovewith the exception that we employed maleic anhydride instead of diethylfumarate in the first step of the synthesis.

Each compound was assayed for the ability to inhibit the formation of P.aeruginosa biofilms. The 2-acetylated compound 10 was inactive in allour assays, providing evidence that the 2-aminoimidazole ring isessential for biological activity. We employed a standard crystal violetreporter assay to assess for the formation of biofilms. Briefly, P.aeruginosa strain PAO1 was allowed to form biofilms in a multi-wellplate in the absence or presence of our compounds. Planktonic (or freegrowing) bacteria were then removed, wells washed vigorously, andcrystal violet added. Crystal violet stains the remaining bacteriawhich, following ethanol solubilization, can be quantitated byspectrophotometry (A₅₄₀). As can be seen from FIG. 1, at 4 hours, bothcompounds inhibited the formation of P. aeruginosa PAO1 biofilms.

Based upon this result, we performed both a time-dependent andconcentration-dependent analysis of each compound. These data aredepicted in FIG. 2. As can be seen, both compounds effectively inhibitformation of P. aeruginosa biofilms at 50 μg/mL (lowest concentrationtested), even at the 48-hour time point (the longest time tested).

To answer the question of whether the compounds were antibiotics or trueinhibitors of biofilm formation, we tested the ability of both compoundsto inhibit growth of planktonic P. aeruginosa. Growth was quantitated bymeasuring optical density at 600 nm. As can be seen in FIG. 3, thecis-compound 11 potently inhibited the growth of planktonic bacteria,indicating that it possesses bactericidal properties and was likelyinhibiting biofilm formation by killing the planktonic bacteria beforebiofilms were established. The trans compound 1, in contrast, marginallyinhibited growth at 24 hours. Therefore, 1 appears to be inhibiting theability of P. aeruginosa to form biofilms.

EXAMPLE 2

Active compounds were tested for inhibition of biofilm formation usingvarious bacterial strains. Results are reported in TABLE 1. Assays wereperformed as reported above, with 500 μM active compound, and 24 hours.“X” denotes biofilm inhibition.

TABLE 1 Formula Compound P. aeruginosa V. Vulnificus H. Pacifica (II)(a)

X (II)(a)

X X X (I)(a)(i)

X X (I)(a)(iv)

X X (I)(a)(xviii)

X X (I)(a)(xviii)

X (I)(a)(xviii)

X (V)

X (I)

X

EXAMPLE 3

Identification of a bicyclic 2-aminoimidazole derivatives that inhibitand disperse bacterial biofilms. In Example 1 above it was demonstratedthe synthesis of a small molecule, denoted TAGE (trans-bromoageliferinanalogue), based on the natural product bromoageliferin, anddemonstrated that TAGE had anti-biofilm activity against Pseudomonasaeruginosa (see FIG. 4). It is demonstrated by the present Example thatTAGE: 1) does not have selective toxicity against cells within thebiofilm state, 2) will inhibit biofilm development under flowconditions, indicating that the CV staining protocol correlates with theability to be active under biomimetic conditions, and 3) TAGE willdisperse preformed P. aeruginosa biofilms. It is also demonstrated thatTAGE is not cytotoxic. Further analogue development (see FIG. 4) hasidentified compounds that are exceedingly effective as biofilminhibitors against the γ-proteobacteria in this study (PAO1, PA14,PDO300, and A. baumannii). Against the β-proteobacterium RB50 and thegram-positive bacterium S. aureus, substantial ability to inhibitbiofilm development is also observed; however, some of this activity isattributed to microbicidal activity. The TAGE derivatives presented inthis study, however, do not disperse pre-formed biofilms with the sameefficiency as TAGE.

The first issue addressed was the ability of TAGE to inhibit theformation of a biofilm from mucoid variants of P. aeruginosa. After a CFpatient is colonized by P. aeruginosa, the bacterium undergoes aphenotypic shift from a non-mucoid to a mucoid form (Govan et al.,Microbiol. Rev., 1996, 60, 539; Ramsey et al., Mol. Microbiol., 2005,56, 309). The mucoid form is characterized by the overproduction ofalginate in the extracellular polymeric substance (EPS) (Ramsey et al.,Mol. Microbiol., 2005, 56, 309). PDO300 was employed to assay if TAGEwould inhibit the formation of mucoid biofilms. PDO300 is awell-characterized mucoid strain of P. aeruginosa that is genotypicallyidentical to PAO1 except for the mucA mutation that converts thebacterium to the mucoid phenotype (Mathee et al., Microbiology, 1999,145, 1349). PDO300 biofilms were allowed to develop in a 96-well platein the absence or presence of TAGE. After 24 hours, the media andplanktonic bacteria were removed, the wells were washed vigorously, andcrystal violet was added. Crystal violet stains the bacterial biofilmthat forms on the inside wall of the well at the air/liquid interface,which, following ethanol solubilization, can be quantitated byspectrophotometry (A₅₄₀) (O'Toole et al., Mol. Microbiol., 1998, 30,295). Using various TAGE concentrations, a dose-response curve of [TAGE]vs. (% biofilm inhibition) was generated and it was determined that TAGEhas an IC₅₀ of 88 μM against PDO300 (FIG. 5). Growth curves (not shown)and colony counts (FIG. 5) indicated that TAGE lacked bactericidalactivity against PDO300.

The second issue addressed was the effect that TAGE had upon thebacterial biofilm. Specifically, the effect TAGE had upon biofilmarchitecture and the effect TAGE had upon cell viability within abiofilm were analyzed. The studies reported above demonstrated that TAGEwas able to significantly reduce biofilm mass at concentrations (300 μM)that induced no microbicidal activity against planktonic bacteria.However, biofilm mass was measured by crystal violet (CV) staining andgives no indication of the topology of the biofilm. CV staining simplyindicates the total amount of biomass that resides within the biofilm.Furthermore, given that there is differential gene expression betweenbacteria growing in a biofilm and planktonic bacteria, (Costerton etal., Science, 1999, 284, 1318.) it was investigated if TAGE hadselective microbicidal activity against biofilm bacteria. To addressthese points, PAO1 biofilms were grown on a glass cover slip for 48hours in the absence or presence of 100 μM TAGE. Media and planktonicbacteria were then washed from the cover slips and live/dead cellstaining was performed. Visualization via confocal microscopy was thenused to assess both the relative ratio of live/dead cells within thebiofilm as well as differences in architecture between the untreated andTAGE-treated samples. A significant reduction in the biofilm biomass andbiofilm architecture was observed with TAGE-treated biofilms incomparison to biofilms grown in the absence of compound (not shown).Furthermore, the ratio of live vs. dead cells in TAGE-treated vs.untreated cells was similar, indicating that TAGE lacks bactericidalactivity against bacteria within a biofilm (not shown).

Next, the ability of TAGE to operate under flow conditions was assessed.Biofilms formed under flow conditions are generally accepted as bettermodels of biofilms that form in vivo. Christensen et al., MethodsEnzymol., 1999, 310, 20. The bacterial strain PAO1:gfp was used toassess biofilm development under flow conditions. PAO1:gfp contains anintegrated green fluorescent protein gene in the PAO1 genome that allowsbacterial visualization via fluorescence. In two separate flow vessels,PAO1:gfp was allowed to attach, under turbulent flow, for 1 hour. It isat this point that irreversible attachment occurs. After the attachmentphase, media only was flowed over one of the vessels whilemedia-containing 300 μM TAGE was flowed over the other vessel. Each flowwas maintained for 24 hours. At the end of 24 hours, each vessel wasvisualized by confocal microscopy. These results clearly show that TAGEis active under continuous flow conditions as evidenced by the severereduction of fluorescence. In addition, the use of the CV staining assaythat was employed in the previous study gave almost identical resultscompared with the results obtained under flow conditions.

Mammalian cytotoxicity of TAGE was also investigated. Bromoageliferin, anatural sponge compound, is known to modulate the activity of calciumchannels. U. Bickmeyer, Toxicon, 2005, 45, 627. GH4Cl rat pituitarycells and N2A mouse neuroblastoma cells were chosen for cytotoxicityscreening. These cell lines are utilized for evaluating toxicity ofmarine natural products. Burkholder et al., Proc. Natl. Acad. Sci. USA,2005, 102, 3471. Van Dolah et al., Nat. Toxins, 1994, 2, 189. Each cellline was plated at 3×10⁴ cells/well in 96-well plates in 50 μl ofDulbeccos Modified Eagles Medium (DMEM). The cells were allowed toadhere at 37° C. in 5% CO₂ for 4 hours before use. 4 μl of testfractions were added and the cells incubated for 18 hours. Cellviability was assessed through an MTT-based colorimetric assay. Allcells remained viable in the presence of up to 600 μM of our compounds,which indicates a lack of cytotoxicity.

One aspect associated with small molecule modulation of biofilmdevelopment is the ability to disperse a pre-formed biofilm. There haveonly been a limited number of examples in the literature of moleculesthat disperse bacterial biofilms. Banin et al., Appl. Environ.Microbiol, 2006, 72, 2064. L. M. Junker and J. Clardy, Antimicrob.Agents Ch., 2007, 51, 3582. Boles et al., Mol. Microbiol., 2005, 57,1210. TAGE was assayed for its ability to disperse pre-formed PAO1 andPA14 biofilms. Each bacterial strain was allowed to form biofilms in amicrotiter well for 24 hours in the absence of compound. At the end of24 hours, the media containing the planktonic bacteria was removed fromeach well and either media alone or media containing TAGE was added.After 24 hours, all the wells were washed vigorously to remove media andplanktonic bacteria, and then were stained with crystal violet toquantify the amount of remaining biofilm. Representative PAO1 resultsare depicted in FIG. 6. TAGE was able to disperse PAO1 with an EC₅₀ of82 μM and PA14 biofilms with an EC₅₀ of 114 μM.

Given the TAGE's ability to inhibit and disperse P. aeruginosa biofilms,the activity of TAGE against other opportunistic bacteria, Acinetobacterbaumannii, Bordetella bronchiseptica, and Staphlococcus aureus, wasinvestigated. A. baumannii is another opportunistic γ-proteobacteriumthat has become a severe threat over the past decade due to itsexceptional and increasing multi-drug resistance. M. E. Falagas and E.A. Karveli, Clin. Microbiol Infect., 2007, 13, 117. B. bronchiseptica isa β-proteobacterium that is frequently isolated from mammalianrespiratory tracts. P. A. Cotter and J. F. Miller, Mol. Microbiol.,1997, 24, 671. S. aureus is a biofilm-forming gram-positive bacteriumthat is frequently associated with nosocomial infections and is becomingan increasing threat due to its acquisition of methicillin andvancomycin resistance. TAGE had no effect on the ability of A.baumannii, B. bronchiseptica, or S. aureus to form biofilms atconcentrations up to 600 μM.

Three TAGE derivatives were synthesized that possessed an acylpyrrolering in a manner that mimics the ageliferin natural products (Scheme 3).

Previous studies have indicated that the incorporation of an acylpyrrole moiety within the 2-aminomidazole scaffold dramaticallyincreasing anti-biofilm activity. These pyrrole 2-aminoimidazolestructures showed anti-biofilm activity that exceeded the anti-biofilmactivity of TAGE and CAGE, while their parent 2-aminoimidazolesubstructure, 4-(3-aminopropyl)-2-aminoimidazole, lacked anti-biofilmactivity. Furthermore, we have also demonstrated that activity is notsolely due to the acyl pyrrole, as simple acyl pyrrole derivatives hadno inhibitory effect on the ability of bacteria to form biofilms. Tosynthesize the three TAGE derivatives, TAGE was first synthesized onmulti-gram scale using the synthetic approach we outlined previous. TAGEwas then coupled to the appropriate the appropriate acyl pyrroletrichloromethyl ketone to generate the 3 TAGE derivatives (PyrroleTAGE,BromoTAGE, and DibromoTAGE).

Screens of the three compounds were performed against A. baumannii andB. bronchiseptica. These strains had showed resistance to TAGE.PyrroleTAGE showed only slight inhibitory activity (23%) against A.baumannii at the highest concentration tested (400 μM). The activity wasencouraging because TAGE showed no activity at more than twice thisconcentration. As noted, incorporation of a bromine atom within the acylpyrrole moiety of the 2-aminoimidazole scaffold might greatly increaseactivity. In this case, BromoTAGE revealed a significant increase inanti-biofilm activity, and it was determined that the IC₅₀ value forinhibition against A. baumannii was 108 μM. DibromoTAGE was shown to beeven a more potent inhibitor against A. baumannii with an IC₅₀ value of15.5 μM. A comparison of the dose-responses for Bromo- and DibromoTAGEis depicted in FIG. 7. These results indicated that the number ofbromines on the acyl pyrrole moieties influenced the inhibitoryactivities of these TAGE derivatives on A. baumannii biofilm formation.Growth curves were measured in the presence or absence of each compound,and it was noted that there was no reduction on planktonic growth (notshown). This indicated that the mechanism of biofilm inhibition withthese TAGE derivatives was not due to microbicidal activity.

Next, the ability of each TAGE derivative to inhibit the formation of B.bronchiseptica biofilms (strain Rb50) was assayed to see if thecompounds could inhibit the formation of biofilms across this bacterialorder. PyrroleTAGE showed no inhibitory activity against Rb50 at 500 μM.However, BromoTAGE showed activity at high concentrations, and furtherinvestigation determined an IC₅₀ value for inhibition of Rb50 to be 385μM. DibromoTAGE was more potent and had an IC₅₀ value of 27 μM(dose-response comparison depicted in FIG. 8). Comparison of the RB50growth curves generated in the absence and presence of the TAGEderivatives, however, revealed that the compounds were microbicidal, andmost likely exerting their anti-biofilm effects through a bacterialstatic mechanism.

Next, the anti-biofilm effects of our TAGE derivatives againstStaphylococcus aureus, a gram-positive bacterium that is known to formbiofilms, were examined. As we observed with TAGE. PyrroleTAGE showed noinhibitory activity at concentrations up to 600 μM. BromoTAGE andDibromoTAGE, however, revealed IC₅₀ values of 21.0 μM and 14.0 μM,respectively (not shown). Analysis of the growth curves generated in theabsence and presence of BromoTAGE and DibromoTAGE revealed that bothcompounds are slightly toxic and have an effect on growth and overallcellular density after 24 hours (not shown). However, these compoundswere not as toxic to S. aureus as they were to RB50.

Given the increase in activity by both BromoTAGE and DibromoTAGE, it wasdetermined if the TAGE derivatives also had enhanced activity against P.aeruginosa. Previous results had shown that TAGE was able to inhibit theformation of PAO1 biofilms with an IC₅₀ of 100 μM. As with all of theprevious studies with PyrroleTAGE, no inhibitory activity was observedat concentrations up to 400 μM. BromoTAGE, however, showed a 3-foldincrease in activity in comparison to TAGE and revealed an IC₅₀ value of32.5 μM against PAO1. Consistent with all of the anti-biofilm resultsfrom this study, DibromoTAGE gave the most significant inhibitionresult, and revealed an IC₅₀ value of 1.77 μM. A representativedose-response comparison is depicted in FIG. 9. Growth curves and colonycounts of PAO1 grown the presence and absence of BromoTAGE andDibromoTAGE indicated that both compounds lacked microbicidal activityagainst planktonic growth. (not shown).

DibromoTAGE was also screened against PDO300 and PA14 to compare to theinhibitory activity of TAGE against these strains. Against PDO300,DibromoTAGE demonstrated enhanced activity with an IC₅₀ value of 2.47μM, while against PA14 DibromoTAGE's IC₅₀ value was determined to be12.0 μM (not shown). In comparison to TAGE, this represents a 35-foldincrease in activity against PDO300 and a 16-fold increase in activityagainst PA14. Growth curves and colony counts demonstrated thatDiBromoTAGE is not toxic to planktonic cells (not shown).

Finally, each of the TAGE derivatives were assayed for their ability todisperse pre-formed biofilms. The increase in anti-biofilm activity thatwe observed for inhibiting the development of bacterial biofilms did nottranslate into increased dispersion activity. At the highestconcentration tested against PAO1 (400 μM), no dispersion was observedby any of the derivatives. Some minimal dispersion activity against A.baumannii biofilms (20-30% at 400 μM) was observed with Bromo- andDiBromoTAGE, but more extensive dispersion was not noted at higherconcentrations. While not wishing to be bound by any particular theory,it is hypothesized that the failure of these derivatives to effectivelydisperse biofilms, in contrast to TAGE, might be due to impededdiffusion through the biofilm matrix.

In conclusion, small molecules were synthesized and studied for theirability to modulate biofilm development. These compounds were shown tobe exceedingly effective as biofilm inhibitors against theγ-proteobacteria in this study (PAO1, PA14, PDO300, and A. baumannii).Against the β-proteobacterium RB50 and the gram-positive bacterium S.aureus, a substantial ability to inhibit biofilm development wasobserved; however microbicidal activity was also observed. In all casesin this study, an increase in activity was correlated to increasedbromine content on the pyrrole moiety. The TAGE derivatives, however, donot disperse pre-formed biofilms with the same efficiency as TAGE. As acore scaffold, it has also been demonstrated that TAGE will inhibitbiofilm development under flow conditions, indicating that the CVstaining protocol correlates with the ability to be active underbiomimetic conditions. Toxicity work indicates that TAGE is devoid ofcytotoxicity, giving these compounds a promising combination of activityand lack of toxicity.

Experimental. All ¹H NMR (300 MHz) and ¹³C NMR (75 MHz) spectra wererecorded at 25.0° C. on a Varian Mercury spectrometer. Chemical shifts(δ) are given in ppm relative to tetramethylsilane or the respective NMRsolvent. Abbreviations used are s=singlet, m=multiplet. High resolutionESI was used to determine molecular weight of new compounds in thisstudy. Silica gel (40 μm average particle size) was used for columnchromatography. All reagents were used without further purification fromcommercial sources unless otherwise noted.

Static Biofilm Inhibition Assay: An overnight culture of the bacterialstrain being screened against was subcultured at an OD₆₀₀ of 0.1 intothe media used (listed below with strain) and then pippetted into testtubes along with a predetermined concentration of our compound ofinterest. Test tubes were then poured into tilted Petri dishes and 100μL of media, strain and compound were then transferred into 96-well PVCmicrotiter plates. These microtiter plates were then covered, wrapped inSaran™ Wrap polyethylene film (S.C. Johnson, Racine, Wis.) and allowedto incubate at 37° C. for 24 hours. After that time, the medium wasdiscarded and the plates were thoroughly washed with water. Theremaining biofilm that was formed during incubation was stained with 100μL of a 0.1% crystal violet solution and allowed to incubate at roomtemperature for 30 minutes. After 30 minutes, the crystal violet wasdiscarded and washed thoroughly again with water. The remaining crystalviolet that had stained the biofilm in the inside walls of the 96-wellPVC plates was solubilized with 200 μL of 95% ethanol. Thequantification of biorilm formation was accomplished by transferring 125μL of the ethanol solution into a polystyrene microtiter dish, which wasread by spectrophotometry (A₅₄₀). After the background has beensubtracted from each row, a percent inhibition can be calculated bydividing the amount of crystal violet in wells that contained compoundby the amount of crystal violet in wells that contained bacteria only.Each concentration reported during the course of this study was repeatedtwo to five times with each of those biofilm inhibition assays beingdone in 6 replicates each.

Bacterial strains/media used: PAO1 - LBNS PA14 - LBNS PDO300 - LBNSAcinetobacter Baumannii (ATCC 19606) - LB Staphylococcus aureus (ATCC29213) - TSB with 0.3% glucose Rb50 - SS media/100X supplement (10 μL/mLof SS media).

Static Biofilm Dispersion Assay: An overnight culture of either PAO1 orPA14 was subcultured at an OD₆₀₀ of 0.5 into LBNS and aliquoted (100 μL)into the wells of a 96-well PVC microtiter plate. The microtiter plateswere then covered and wrapped with Saran™ Wrap polyethylene film beforebeing incubated at room temperature under stationary conditions for 24hours to allow the formation of biofilms. After 24 hours, the medium wasdiscarded and the plates were thoroughly washed with water leaving onlythe preformed biofilm on the inside of the PVC wells. LBNS alone wasadded for the control and LBNS containing a predetermined concentrationof the compound of interest was then pippetted into the wells (100 μL).The plates were then covered and wrapped in Saran™ Wrap polyethylenefilm and incubated at 37° C. for an additional 24 hours after which themedia and planktonic cells were discarded and washed with water. Thewells were then stained with 100 μL of a 0.1% crystal violet solutionand allowed to sit for 30 minutes at room temperature. The crystalviolet solution was then discarded from the wells and washed with waterthoroughly. The remaining crystal violet was solubilized with 200 μL of95% ethanol. The quantification of remaining biofilm was accomplished bytransferring 125 μL of the ethanol solution into a polystyrenemicrotiter dish, which was read by spectrophotometry (A₅₄₀). Percentdispersion was calculated in the same manner as percent biofilminhibition described previously in this experimental section.

General Procedure for Acylation of TAGE. DMF (2 or 3 ml) was added to areaction vial containing TAGE•3 HCl along with Na₂CO₃ (5 equivalents),and the respective brominated or non-brominated2-(trichloroacetyl)-pyrrole variant (2.1 equivalents). The reaction wasthen allowed to stir under an argon atmosphere at 50° C. overnight. Uponcompletion of the reaction, the reaction vial was removed from the heatsource and concentrated under reduced pressure vacuum. The resultingresidue was purified by flash column chromatography (utilizing agradient starting at 10% methanol/ammonia in DCM and increasing polarityto 40% methanol/ammonia in DCM) to give the corresponding acylpyrroleTAGE derivative. Percent yields are recorded with compoundcharacterization below.

PyrroleTAGE—DMF (2 ml), 104.4 mg TAGE•3 HCl, 155.6 mg2-(trichloroacetyl)-pyrrole, 183 mg Na₂CO₃ yielded 69 mg PyrroleTAGE asa free base after purification (53% yield). ¹H NMR (300 Hz, DMSO-d₆)δ11.47 (s, 2H), δ 8.20 (s, 2H), δ 7.40 (s, 2H), δ 6.84 (s, 2H), δ 6.77(s, 2H), δ 6.07 (s, 2H), δ 3.21 (m, 4H), δ 2.62-2.50 (m, 2H partiallyburied in DMSO peak), δ 2.26-2.16 (m, 4H) ppm; ¹³C NMR (75 Hz, DMSO-d₆)δ 160.8, 146.5, 126.3, 121.1, 117.6, 110.2, 108.5, 40.7, 34.3, 20.1;HRMS (ESI) calcd for C₁₉H₂₄N₇O₂ (MH)⁺382.1985, found 382.1982.

BromoTAGE—DMF (2 ml), 101 mg TAGE•3 HCl, 210.7 mg4-bromo-2-(trichloroacetyl)-pyrrole, 180 mg Na₂CO₃ yielded 89 mgBromoTAGE as a free base after purification (50% yield). ¹H NMR (300 Hz,DMSO-d₆) δ11.91 (s, 2H), δ 8.44 (t, 2H), δ 7.42 (s, 2H), δ 6.95 (s, 2H),δ 6.91 (s, 2H), δ 3.15 (m, 4H), δ 2.60-2.50 (m, 2H partially buried inDMSO peak), δ 2.25-2.16 (m, 4H) ppm; ¹³C NMR (75 Hz, DMSO-d₆) δ 159.7,146.5, 126.9, 121.0, 117.5, 111.9, 94.9, 40.8, 34.0, 20.1; HRMS (EST)calcd for C₁₉H₂₂N₇O₂Br₂ (MH)⁺538.0196, found 538.0185.

DibromoTAGE—DMF (3 ml), 106 mg TAGE•3 HCl, 274 mg 4,5-dibromo-2-(trichloroacetyl)-pyrrole, 195 mg Na₂CO₃ yielded 127 mgDibromoTAGE as a free base after purification (52% yield). ¹H NMR (300Hz, DMSO-d₆) δ 8.51 (t, 2H), δ 7.41 (s, 2H), δ 6.98 (s, 2H), δ 3.19 (m,4H), δ 2.60-2.50 (m, 2H partially buried in DMSO peak), δ 2.25-2.16 (m,4H) ppm; ¹³C NMR (75 Hz, DMSO-d₆) δ 158.9, 146.5, 128.1, 117.5, 113.1,104.3, 97.8, 40.8, 33.9, 20.1; HRMS (ESI) calcd for C₁₉H₂₀N₇O₂Br₄(MH)⁺693.8406, found 693.8410.

EXAMPLE 4

Inhibition and dispersion of Pseudomonas aeruginosa biofilms withreverse amide 2-aminoimidazole oroidin analogues. The marine alkaloidoroidin along with a small library of reverse amide (RA)2-aminoimidazoles were synthesized and assayed for anti-biofilm activityagainst PAO1 and PA14, two strains of the medically relevantγ-proteobacterium Pseudomonas aeruginosa. Analogues that contained along, linear alkyl chain were more potent inhibitors than the naturalproduct at preventing the formation of PAO1 and PA14 biofilms. The mostactive compound in the series was also shown to disperse establishedPAO1 and PA14 biofilms at low micromolar concentrations.

The activity of oroidin, a natural sponge compound, has been documentedin a limited number of studies involving bacterial attachment andcolonization. Kelly et al., Aquat. Microb. Ecol., 2005, 40, 191-203.Kelly et al., Aquat. Microb. Ecol., 2003, 31, 175-182. Oroidin has alsobeen shown to inhibit biofouling driven by the marine α-proteobacteriumR. salexigens. A. Yamada et al., Bull. Chem. Soc. Jpn., 1997, 70,3061-3069. Due to this activity and chemical simplicity, oroidin wasselected as a lead compound for structure activity relationship (SAR)studies in hopes of discovering a diverse range of compounds thatpossess anti-biofilm properties. One intriguing approach is the reversalof the amide bond highlighted in FIG. 10, which connects thebromopyrrole tail of oroidin 4 to the 2-aminoimidazole (2-AI) head.

One of the best methods for large scale preparation of the 2-AI scaffolden route to prepare oroidin and other family members involves Akaborireduction (Na/Hg) of ornithine methyl ester 7 followed by condensationwith cyanamide under pH controlled conditions. S. Akabori, Ber. Dtsch.Chem. Ges., 1933, 66, 151-158. G. C. Lancini and E. Lazzari, J.Heterocycl. Chem., 1966, 3, 152-&. A. Olofson et al., J. Org. Chem.,1998, 63, 1248-1253; Oroidin was synthesized as reported and matchedcharacterization data. Derivatization can then be achieved via acylationof the alkyl amine off the carbon tail with variously substitutedtrichloroacetyl pyrroles. However, this chemistry is plagued by severelimitations, most notably the overall lack of compatibility of thissystem with other trichloroacetyl esters. In addition, solubility issuesof the parent 2-AI leaves much to be desired. Many attempts by our groupin developing other acylation conditions that would allow for thegeneration of greater diversity have proven unfruitful. From a practicalstandpoint, purifications of intermediates bearing an unprotected 2-AIoften require large amounts of methanol saturated with ammonia(MeOH/NH3), which is cumbersome to prepare and can be difficult toremove from the pure sample after column chromatography.

Implementation of a reverse amide approach, coupled with a practicalprotecting group strategy, would effectively eliminate many of theaforementioned handicaps with current methods. Installation of thereverse amide bond could be obtained by direct aminolysis of anintermediate Boc-2AI alkyl ester or through couplings of a carboxylicacid (FIG. 10). These intermediates could be accessed throughα-bromoketones which are obtained by diazomethane homologation with theproper acyl chloride. Additionally, significant diversity can beachieved by incorporating any commercially available amine with a commonRA intermediate. Herein we report the synthesis of a focused reverseamide (RA) library (FIG. 11) and subsequent biological evaluation of thelibrary in comparison to the natural product oroidin in the context ofanti-biofilm activity of biofilms formed by the medically relevantγ-proteobacterium Pseudomonas aeruginosa.

Synthesis of RA Library. Scaffold synthesis began with treatment of thecommercially available acid chloride 17 with diazomethane (Scheme 4).Adiyaman et al., Tet. Lett., 1996, 37, 4849-4852. Quenching withconcentrated HCl or HBr delivered the corresponding α-haloketones inexcellent yields which were isolable by column chromatography.Installation of the protected 2-aminoimidazole moiety was achievedthrough a Boc-guanidine condensation in DMF at ambient temperature toyield 18. Significantly higher yields for this step were obtained whentwo equivalents of sodium iodide were added to the reaction mixture andrepresents a significant improvement over previous reports. N. Ando andS. Terashima, Synlett, 2006, 2836-2840. V. B. Birman and X. T. Jiang,Org. Lett., 2004, 6, 2369-2371. It was also observed during thissequence that the α-bromoketone afforded higher yields than its α-chlorocounterpart in the cyclization reaction.

The first approach to the RA scaffold relied heavily on the aminiolysisof intermediate 18 since this would afford the Boc-protected RAprecursors in a single synthetic step. After deprotection with TFA andHCl salt exchange, isolation of the targets would require onlyfiltration with no need for further purification. Based upon the seminalpaper published by Weinreb on the transformation, trimethylaluminum wasused as the Lewis acid to affect the direct aminolysis reaction. Bashaet al., Tet. Lett., 1977, 4171-4174. Numerous reaction factors weretaken into account such as choice of solvent, equivalents ofaluminum-amine complex, reagent order of addition, time, andtemperature. Despite all of the conditions scanned (data not shown), thehighest yielding reaction occurred in only 55% yield when aniline wasused as the amine partner. Triazabicyclo[4.4.0]dec-5-ene (TBD) was alsoexamined as a potential catalyst to promote the direct aminolysis ofester 18. Sabot et al., Tet. Lett., 2007, 48, 3863-3866. Heating bothstarting materials in the presence of 30 mol % of TBD in toluene atelevated temperatures for extended periods of time failed to produce anydesired product as evident by TLC analysis (data not shown).

Due to the problems encountered utilizing aminolysis, we opted for amore conventional route to access the RA scaffold through theintermediacy of an activated carboxylic acid. Unfortunately,saponification of the methyl ester 18 proved problematic on this systemas cleavage of the Boc group was observed under the basic conditions ofboth LiOH/MeOH/THF/H₂O or LiI/pyridine. Decomposition of the methylester was also observed when TMSOK in methylene chloride or (Bu₃Sn)₂O intoluene at either ambient temperature or reflux were employed as thesaponification agents (data not shown).

Persuaded by these results that the current route required revision, webegan a second generation approach to our core scaffold (Scheme 5). Thisapproach relied on a different protecting group strategy, substitutingthe methyl ester for a benzyl ester which, in the case of another failedattempt at aminolysis, would undergo hydrogenolysis under mildconditions to deliver the corresponding Boc-protected acid 22. Synthesisbegan with the known mono benzyl ester acid (Li et al., J. Am. Chem.Soc., 1995, 117, 2123-2127.20 which was transformed into the benzylprotected α-bromoketone by conversion to its acid chloride followed bydiazomethane homologation and concomitant quench with concentrated HBr.Cyclization of this intermediate afforded the Boc-protected 2-AI 21 in66% yield. All attempts at direct aminolysis of benzyl ester 21 resultedin sluggish reactions that were plagued by the formation of multipleside products.

Given the failure of the direct aminolysis conversion, the two-stepapproach to the RA scaffold was investigated. Deprotection proceeded asplanned and was accomplished by subjecting 21 to a hydrogen atmosphereat balloon pressure to cleanly afford pure Boc-protected acid 22 in nearquantitative yield (98%). With the acid now in hand and available on amulti-gram scale, attempts to install the key amide bond were assessed.A number of activating agents were scanned including DCC, EDC, HCTU,CDI, and cyanuric chloride to affect the transformation. Of those listedonly EDC and HCTU were able to give consistent and tangible results. EDCwas chosen over HCTU due to ease of purification in separating sideproducts during column chromatography. It was during this optimizationthat the limitation of the synthetic route was identified to be thereactivity of the Boc group. A significant quantity of a Boc-protectedstarting amine was isolated and characterized, signifying the labilityof the Boc-group due to Boc-transfer under the reaction conditionsregardless of which activating agent was used.

With two routes in hand to generate the RA scaffold, we assembled thefocused library outlined in Table 2. EDC/HOBt couplings of acid 22 wereused to generate most of the linear alkyl chain analogues (28-34%) whileaminolysis of the methyl ester intermediate 18 furnished the remainingcompounds (11-55%) in the library (Table 2). The final step of thesynthetic approach required removal of the Boc group, which proceeded atroom temperature in TFA/DCM. The resulting trinflouroacetate salts ofeach target were then traded out for their HCl counterparts beforecharacterization and assessment of their biological activity.

TABLE 2 Completion of the reverse amide library

Coupled Amine Conditions Product Target isobutylamine a 23 8 hexylamineb 24 9 octylamine b 25 10 decylamine a 26 11 dododecylamine b 27 12cyclopentylamine a 28 13 morpholine a 29 14 aniline a 30 152-aminopyrimidine a 31 16 Reaction conditions: (a) AlMe₃, 18, DCE, 0° C.to 60° C. (b) 22, EDC, HOBt, DMF (c) TFA, CH₂Cl₂ (d) 2M HCl in Et₂O

Biological Evaluation of RA Library. Nosocomial infections are driven bya persistent bacterial colonization of hospital facilities, wherein thebacteria are extremely resistant to eradication because they exist in abiofilm state. P. aeruginosa is an opportunistic γ-proteobacterium thatis a serious threat to immunocompromised patients and is frequentlyisolated from patients found in intensive care units suffering fromsevere burns or other traumas. It is the second most common pathogen inhospital-acquired pneumonia behind Staphylococcus aureus. Driscoll etal., Drugs, 2007, 67, 351-368. For Cystic Fibrosis patients, the onsetof colonization by this bacterium is of great concern. Morbidity ratesof patients who suffer from the disease are directly correlated to thevirulence of P. aeruginosa biofilms. J. W. Costerton, Trends inMicrobiology, 2001, 9, 50-52. T. F. C. Mah and G. A. O'Toole, TrendsMicro., 2001, 9, 34-39. The speed and prevalence with which multidrugresistant (MDR) strains are appearing puts pressure on the medicalcommunity to find ways to combat the aggressive nature of thisbacterium. M. E. Falagas and E. A. Karveli, Clin. Microbiol. Infec.,2007, 13, 117-119.

Members of the reverse amide library along with oroidin Olofson et al.,J. Org. Chem., 1998, 63, 1248-1253; Oroidin was synthesized as reportedand matched characterization data. were initially screened at 500 μM ina 96-well format using a crystal violet reporter assay to assess eachcompound's ability to inhibit the formation of PAO1 or PA14 biofilms(FIG. 12). G. A. O'Toole and R. Kolter, Mol. Microbiol., 1998, 28,449-461. Compounds 5, 19, and 32 were used as controls in the assays andall showed only marginal inhibition. There was a remarkable range ofactivities among the RA compounds analyzed in the inhibition assay.Similar activities were observed between PAO1 and PA14, although mostcompounds were slightly more potent against PA14. This trend is oppositeto our previously reported bromoageliferin analogues. Huigens et al., J.Am. Chem. Soc., 2007, 129, 6966-6967. These screens also suggested thatthe aliphatic chain derivatives (9-12) and oroidin 4 were very potentinhibitors of P. aeruginosa biofilms.

Subsequently, the aliphatic derivatives (9-12) and oroidin 4 wereselected for IC₅₀ value determination against PAO1 and PA14 (Table 3).The generation of dose-response curves for these compounds revealed acorrelation between the length of the carbon chain and the potency ofthe compound (supporting information). This trend is apparent when theIC₅₀ values are plotted as a function of chain length in both PAO1 andPA14 (FIG. 13). Increasing the chain length from six to twelve carbonseffectively increased the inhibition activity over a full order ofmagnitude in both strains. All linear carbon chain analogues weresignificantly more potent than oroidin (PAO1 IC₅₀=190 μM, PA14 IC₅₀=166μM). The most active RA analogue identified was 12 (PAO1 IC₅₀=2.84 μM,PA14 IC₅₀=2.26 μM), effectively demonstrating that changes to certainportions of the natural product have the ability to dramaticallyincrease biological activity.

TABLE 3 PAO1 and PA14 IC₅₀ values. Compound PAO1 IC₅₀ (μM) PA14 IC₅₀(μM) oroidin (4) 190 ± 9  166 ± 23   9 (n = 5) 32.7 ± 6.5  39.9 ± 13.010 (n = 7) 18.4 ± 2.3  13.3 ± 1.8  11 (n = 9) 7.79 ± 1.52 7.48 ± 1.60 12(n = 11) 2.84 ± 0.93 2.26 ± 0.83

To validate that our compounds were true inhibitors of biofilm formationand not acting as bactericidal agents, growth curves were performed atthe determined IC₅₀ values for PAO1 and PA14 with the dodecyl-basedanalogue 12 and oroidin 4. Bacterial cell densities for both strainsremained unchanged when grown in the presence or absence of either thenatural product 4 or 12 throughout a 24-hour time period (supportinginformation).

While the focus has predominantly been on designing small molecules thatinhibit the formation of biofilms, the more significant challenge is thedevelopment of a small molecule that will disperse established biofilms.Treatment of chronic infections is commonly hindered by the presence ofestablished biofilms that impart increased resistance to conventionalantibiotics. Costerton et al., Science, 1999, 284, 1318-1322. Smallmolecules able to disperse established biofilms are, therefore, of greatinterest to the medical community. To test for the ability to disperseestablished biofilms, PAO1 and PA14 were allowed to form biofilms for 24hours in the absence of compound. After this time the media wasdiscarded. The wells were washed and fresh media was added containingvarying concentrations of 12 and then incubated at 37° C. for 24 hours.RA analogue 12 displayed significant anti-biofilm activity, dispersingestablished PAO1 and PA14 biofilms with EC₅₀ values of 32.8±4.7 μM and21.3±3.9 μM respectively (FIG. 14).

In conclusion, we have identified several reverse amide (RA) analoguesthat possess potent anti-biofilm properties. These compounds are basedon a reverse amide scaffold which switches the directionality of theamide bond frequently found in many members of the oroidin class ofmarine alkaloids. The synthetic path taken to access these derivativesallows for rapid access and simplified purification of all libraryanalogues. Clearly, the most potent derivatives were those thatcontained linear carbon chains of various lengths from the amidenitrogen. The most active of these compounds, 12, has also been shown todisperse established P. aeruginosa biofilms at low micromolarconcentrations, making it a highly noteworthy addition to the limitednumber of small molecules known to possess such characteristics. L. M.Junker and J. Clardy, Antimicrob. Agents Ch., 2007, 51, 3582-3590. Baninet al., Appl. Environ. Microb., 2006, 72, 2064-2069. Boles et al., Mol.Microbiol., 2005, 57, 1210-1223.

Experimental. Stock solutions (100, 10, 1 mM) of all compounds assayedfor biological activity were prepared in DMSO and stored at roomtemperature. The amount of DMSO used in both inhibition and dispersionscreens did not exceed 1% (by volume). P. aeruginosa strains PAO1 andPA14 were graciously supplied by the Wozniak group at Wake ForestUniversity School of Medicine.

General Static Inhibition Assay Protocol for Pseudomonas aeruginosa. Anovernight culture of the wild type strain was subcultured at an OD₆₀₀ of0.10 into LBNS along with a predetermined concentration of the smallmolecule to be tested for biofilm inhibition. Samples were thenaliquoted (100 μL) into the wells of a 96-well PVC microtiter plate. Themicrotiter dishes were covered and sealed before incubation understationary conditions at 37° C. for 24 hours. After that time, themedium was discarded and the plates thoroughly washed with water. Thewells were then inoculated with a 0.1% aqueous solution of crystalviolet (100 μL) and allowed to stand at ambient temperature for 30minutes. Following another thorough washing with water the remainingstain was solubilized with 200 μL of 95% ethanol. Biofilm inhibition wasquantitated by measuring the OD₅₄₀ for each well by transferring 125 μLof the ethanol solution into a fresh polystyrene microtiter dish foranalysis.

General Static Dispersion Assay Protocols for Pseudomonas aeruginosa. Anovernight culture of the wild type strain was subcultured at an OD₆₀₀ of0.50 into LBNS and then aliquoted (100 μL) into the wells of a 96-wellPVC microtiter plate. The microtiter dishes were covered and sealedbefore incubation under stationary conditions at room temperature toallow formation of the biofilms. After 24 hours the medium was discardedand the plates thoroughly washed with water. Fresh medium containing theappropriate concentration of compound was then added to the wells. Theplates were again sealed and this time incubated under stationaryconditions at 37° C. After 24 hours, the media was discarded from thewells and the plates washed thoroughly with water. The wells wereinoculated with a 0.1% aqueous solution of crystal violet (100 μL) andallowed to stand at ambient temperature for 30 minutes. Followinganother thorough washing with water the remaining stain was solubilizedwith 200 μL of 95% ethanol. Biofilm dispersion was quantitated bymeasuring the OD₅₄₀ for each well by transferring 125 μL of the ethanolsolution into a fresh polystyrene microtiter dish for analysis.

Chemistry. All reagents including anhydrous solvents used for thechemical synthesis of the library were purchased from commerciallyavailable sources and used without further purification unless otherwisenoted. All reactions were run under either a nitrogen or argonatmosphere. Flash silica gel chromatography was performed with 60 Å meshstandard grade silica gel from Sorbtech. ¹H and ¹³C NMR spectra wereobtained using Varian 300 MHz or 400 MHz spectrometers. NMR solventswere purchased from Cambridge Isotope Labs and used as is. Chemicalshifts are given in parts per million relative to DMSO-d₆ (δ 2.50) andCDCl₃ (δ 7.27) for proton spectra and relative to DMSO-d₆ (δ 39.51) andCDCl₃ (δ 77.21) for carbon spectra with an internal TMS standard.High-resolution mass spectra were obtained at the North Carolina StateMass Spectrometry Laboratory for Biotechnology. ESI experiments werecarried out on Agilent LC-TOF mass spectrometer.

6-bromo-5-oxo-hexanoic acid methyl ester. Methyl glutaryl chloride (2.5mL, 18.23 mmol) was dissolved into anhydrous dichloromethane (10 mL) andadded drop-wise to a 0° C. solution of CH₂N₂ (55.0 mmol generated fromDiazald® diazomethane precursor/KOH) in diethyl ether (150 mL). Thissolution was stirred at 0° C. for 1.5 h at which time the reaction wasquenched via the drop-wise addition of 48% HBr (7.5 mL). The reactionmixture was diluted with dichloromethane (25 mL) and immediately washedwith sat. NaHCO₃ (3×25 mL) and brine (2×25 mL) before being dried(MgSO₄), filtered and concentrated. The crude oil was purified via flashcolumn chromatography (10-30% EtOAc/Hexanes) to obtain the titlecompound (3.76 g, 93%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ3.91 (s, 2H), 3.68 (s, 3H), 2.76 (t, 2H, J=7.2 Hz), 2.38 (t, 2H, J=7.2Hz), 1.95 (quint, 2H, J=7.2 Hz); ¹³C NMR (75 MHz, CDCl₃) δ 201.36,173.41, 51.67, 38.74, 34.16, 32.87, 19.13; HRMS (ESI) calcd forC₇H₁₂O₃Br (MH)⁺222.9964, found 222.9964.

6-chloro-5-oxo-hexanoic acid methyl ester. Using the same generalprocedure as used above but instead quenching with conc. HCl affordedthe chloro derivative (2.93 g, 90%) as a colorless oil. ¹H NMR (400 MHz,CDCl₃) δ 4.13 (s, 2H), 3.67 (s, 3H), 2.69 (t, 2H, J=7.2 Hz), 2.38 (t,2H, J=7.2 Hz), 1.94 (quint., 2H, J=7.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ201.85, 173.33, 51.57, 48.22, 38.47, 32.66, 18.61; HRMS (ESI) calcd forC₇H₁₂O₃Cl (MH)⁺179.0469, found 179.0476.

6-bromo-5-oxo-hexanoic acid benzyl ester. Monobezylesterbutanoic acid 20(3.00 g, 13.6 mmol) was dissolved in anhydrous dichloromethane (70 mL)at 0° C. and a catalytic amount of DMF was added. To this solution wasadded oxalyl chloride (3.60 mL, 41.3 mmol) drop-wise and the solutionwas then warmed to room temperature. After 1 h, the solvent and excessoxalyl chloride were removed under reduced pressure. The resulting solidwas dissolved into anhydrous dichloromethane (10 mL) and added drop-wiseto a 0° C. solution of CH₂N₂ (42.0 mmol generated from Diazald®diazomethane precursor (Sigma-Aldrich, St. Louis, Mo.)/KOH) in diethylether (120 mL). This solution was stirred at 0° C. for 1.5 h at whichtime the reaction was quenched via the drop-wise addition of 48% HBr(4.7 mL). The reaction mixture was diluted with dichloromethane (25 mL)and immediately washed with sat. NaHCO₃ (3×25 mL) and brine (2×25 mL)before being dried (MgSO₄), filtered and concentrated. The crude oil waspurified by flash column chromatography (0-30% EtOAc/Hexanes) to obtainthe title compound (3.57 g, 88%) as a colorless oil. ¹H NMR (400 MHz,CDCl₃) δ 7.35 (m, 5H), 5.12 (s, 2H), 3.85 (s, 2H), 2.73 (t, 2H, J=6.8Hz), 2.42 (t, 2H, J=6.8 Hz), 1.96 (quint, 2H, J=6.8 Hz); ¹³C NMR (75MHz, CDCl₃) δ 201.43, 172.88, 136.15, 128.79, 128.48, 128.45, 66.52,38.74, 34.07, 33.20, 19.23; HRMS (ESI) calcd for C₁₃H₁₆O₃Br (MH)³⁰299.0277, found 299.0279.

2-amino-4-(3-methoxycarbonyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (18). 6-bromo-5-oxo-hexanoic acid methyl ester (2.30 g,10.3 mmol), Boc-guanidine (4.92 g, 30.9 mmol),³² and Nal (3.07 g, 20.6mmol) were dissolved in DMF (30 mL) and allowed to stir at roomtemperature. After 24 h the DMF was removed under reduced pressure andthe residue was taken up in ethyl acetate (100 mL) and washed with water(3×50 mL) and brine (50 mL) before being dried (Na₂SO₄), filtered andevaporated to dryness. The resulting oil was purified by flash columnchromatography (50-100% EtOAc/Hexanes) to obtain a yellow oil.Trituration of the viscous oil with cold hexanes (20 mL) produced aprecipitate, which upon filtration yielded 18 (1.89 g, 65%) as a paleyellow solid. ¹H NMR (400 MHz, CDCl₃) δ 6.53 (s, 1H), 5.6 (br s, 2H),2.41 (t, 2H, J=7.2 Hz), 2.37 (t, 2H, J=7.2 Hz), 1.93 (quint., 2H, J=7.2Hz), 1.58 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 174.09, 150.11, 149.61,138.39, 107.15, 84.81, 51.56, 33.65, 28.18, 27.68, 23.82; HRMS (ESI)calcd for C₁₃H₂₂N₃O₄ (MH)⁺284.1604, found 284.1606.

4-(2-amino-1H-imidazol-4-yl) butyric acid hydrochloride (19). To2-amino-4-(3-methoxycarbonyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester 18 (50 mg, 0.176 mmol) was added methanol (0.60 mL),tetrahydrofuran (0.20 mL), and water (0.20 mL). Lithium hydroxide (9 mg,0.352 mmol) was then added and the reaction was stirred at roomtemperature for 30 min. The pH of the solution was carefully adjusted topH=5 with a 1N aqueous solution of HCl before being evaporated todryness. The crude product was purified via a silica gel plug (100% MeOHsat. NH₃) to deliver the product as its corresponding free base. Thehydrochloride salt was obtained through addition of a single drop ofconcentrated HCl to a methanolic solution (2 mL) of the free base.Rotary evaporation of this solution afforded 19 (34 mg, 94%) as a whitesolid. ¹H NMR (300 MHz, DMSO-d₆) δ 12.25 (s, 1H), 12.13 (br s, 1H),11.77 (s, 1H), 7.33 (s, 2H), 6.54 (s, 1H) 2.43 (t, 2H, J=7.2 Hz), 2.21(t, 2H, J=7.2 Hz), 1.73 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 174.04,146.92, 126.01, 108.62, 32.81, 23.47, 23.05; HRMS (ESI) calcd forC₇H₁₂N₃O₂ (MH)⁺170.0924, found 170.0927.

2-amino-4-(3-benzyloxycarbonyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (21). 6-bromo-5-oxo-hexanoic acid benzyl ester (3.42 g,11.99 mmol) and Boc-guanidine (5.73 g, 35.97 mmol) were dissolved in DMF(35 mL) and allowed to stir at room temperature. After 48 h the DMF wasremoved under reduced pressure and the residue was taken up in ethylacetate (100 mL) and washed with water (3×50 mL) and brine (50 mL)before being dried (Na₂SO₄), filtered and evaporated to dryness. Theresulting oil was purified by flash column chromatography (30-100%EtOAc/Hexanes) to obtain the title compound (2.79 g, 66%) as a colorlessoil which solidified upon prolonged standing. ¹H NMR (400 MHz, CDCl₃) δ7.35 (m, 5H), 6.51 (s, 1H), 5.91 (s, 2H), 5.12 (s, 2H), 2.41 (m, 4H),1.94 (quint., 2H, J=7.2 Hz), 1.57 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ173.45, 150.31, 149.59, 138.27, 136.36, 128.67, 128.29, 128.27, 107.05,84.73, 66.23, 33.82, 28.16, 27.62, 23.79; HRMS (ESI) calcd forC₁₉H₂₆N₃O₄ (MH)⁺360.1917, found 360.1919.

2-amino-4-(3-carboxy-propyl)-imidazole-1-carboxylic acid tert-butylester (22). To a solution of anhydrous THF (2 mL) and 10% Pd/C (12 mg)was charged2-amino-4-(3-benzyloxycarbonyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester 21 (101 mg, 0.281 mmol). Air was removed from thesystem and the reaction was back flushed with hydrogen. This process wasrepeated three times before setting the reaction under a hydrogenballoon at atmospheric pressure and temperature for 1 h. After that timethe reaction was filtered through a Celite® diatomite pad (WorldMinerals Inc., Santa Barbara, Calif.) and the filter cake was washedwith THF (8 mL). The filtrate was concentrated under reduced pressure toafford the title compound 21 (75 mg, 98%) as a white solid. ¹H NMR (400MHz, DMSO-d₆) δ 6.52 (s, 1H), 6.42 (br s, 2H), 2.52 (t, 2H, J =5.4 Hz),2.18 (t, 2H, J=5.4 Hz), 1.71 (m, 2H), 1.53 (s, 9H); ¹³C NMR (100 MHz,DMSO-d₆) δ 175.00, 149.99, 148.95, 138.28, 105.86, 84.09, 39.24, 38.85,33.70, 27.52, 27.08, 23.52; HRMS (ESI) calcd for C₁₂H₂₀N₃O₄(MH)⁺270.1448, found 270.1452.

General aminolysis procedure: To a stirring 0° C. solution of amine(0.704 mmol) in anhydrous 1,2-dichloroethane (1 mL) was added drop-wisea 2M solution of AlMe₃ in PhCH₃ (0.351 mL, 0.704 mmol). The solution wasstirred for 10 min before the addition of2-amino-4-(3-methoxycarbonyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester 18 (100 mg, 0.352 mmol) in several portions. Oncedissolution was complete, the reaction was warmed to 60° C. and stirreduntil completion as evident by TLC analysis. The reaction was thencooled back down to 0° C. before being diluted with dichloromethane (5mL) and quenched with water (1 mL). The resulting viscous solution waswarmed to ambient temperature and Celite® diatomite was added. Afterstirring for 5 min, the mixture was filtered and the filtrate washedwith brine (2×3 mL), dried (Na₂SO₄), and evaporated to dryness. Thecrude product was purified via flash column chromatography (2-10%MeOH/CH₂Cl₂) to afford pure product.

2-amino-4-(3-isobutylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (23) White solid (46 mg, 40%). ¹H NMR (400 MHz,DMSO-d₆) δ 7.75 (m, 1H), 6.51 (s, 1H), 6.37 (br s, 2H), 2.85 (t, 2H,J=6.4 Hz), 2.22 (t, 2H, J=6.8 Hz), 2.07 (t, 2H, J=7.2 Hz), 1.61-1.73 (m,3H), 1.53 (s, 9H), 0.81 (d, 6H, J=6.4 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ171.82, 149.92, 148.95, 138.39, 105.84, 84.04, 45.94, 34.88, 28.09,27.52, 27.23, 24.10, 20.14; HRMS (EST) calcd for C₁₆H₂₉N₄O₃(MH)⁻325.2234, found 325.2238.

2-amino-4-(3-decylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (26). Tan solid (24 mg, 16%). ¹H NMR (400 MHz, DMSO-d₆)δ 7.74 (m, 1H), 6.52 (s, 1H), 6.49 (br s, 2H), 3.00 (q, 2H, J=6.8 Hz),2.22 (t, 2H, J=6.8 Hz), 2.04 (t, 2H, J=6.8 Hz), 1.71 (quint., 2H, J=6.8Hz), 1.53 (s, 9H), 1.36 (m, 2H), 1.23 (s, 14H), 0.85 (t, 3H, J=6.8 Hz);¹³C NMR (100 MHz, DMSO-d₆) δ 171.64, 149.82, 148.86, 137.90, 105.90,84.16, 38.33, 34.88, 31.31, 29.16, 29.03, 28.97, 28.75, 28.73, 28.02,27.51, 27.05, 26.40, 24.04, 22.12, 13.97; HRMS (ESI) calcd forC₂₂H₄₁N₄O₃ (MH)⁺409.3173, found 409.3175.

2-amino-4-(3-cyclopentylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (28). White solid (54 mg, 45%). ¹H NMR (400 MHz,DMSO-d₆) δ 7.72 (d, 1H, J=6.4 Hz), 6.5 (s, 1H), 6.38 (s, 2H), 3.97 (m,1H), 2.21 (t, 2H, J=7.2 Hz), 2.02 (t, 2H, J=7.2 Hz), 1.73 (m, 4H), 1.61(m, 2H), 1.53 (s, 9H), 1.47 (m, 2H), 1.32 (m, 2H); ¹³C NMR (75 MHz,DMSO-d₆) δ 171.27, 149.92, 148.96, 138.37, 105.88, 84.02, 50.04, 34.79,32.32, 28.05, 27.53, 27.17, 24.00, 23.43; HRMS (ESI) calcd forC₁₇H₂₉N₄O₃ (MH)⁺337.2234, found 337.2235.

2-amino-4-(4-morpholin-4-yl-4-oxo-butyl)-imidazole-1-carboxylic acidtert-butyl ester (29). Tan solid (33 mg, 27%). ¹H NMR (400 MHz, DMSO-d₆)δ 6.52 (s, 1H), 6.39 (s, 2H), 3.52 (m, 4H), 3.41 (m, 4H), 2.28 (m, 4H),1.42 (quint., 2H, J=7.2 Hz) 1.53 (s, 9H); ¹³C NMR (75 MHz, DMSO-d₆) δ171.43, 150.56, 149.64, 139.17, 106.58, 84.78, 66.85, 46.14, 32.23,28.23, 27.89, 24.19; HRMS (ESI) calcd for C₁₆H₂₇N₄O₄ (MH)⁺339.2026.found 339.2027.

2-amino-4-(3-phenylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (30). White solid (66 mg, 55%). ¹H NMR (300 MHz,DMSO-d₆) δ 9.88 (s, 1H), 7.59 (d, 2H, J=8.1 Hz), 7.27 (t, 2H, J=7.5 Hz),7.00 (t, 1H, J=7.2 Hz), 6.55 (s, 1H), 6.44 (br s, 2H), 2.97 (m, 4H),1.82 (m, 2H), 1.53 (s, 9H); ¹³C NMR (75 MHz, DMSO-d₆) δ 170.95, 149.71,148.86, 139.25, 138.16, 128.47, 122.78, 118.98, 105.92, 84.00, 38.42,35.71, 27.44, 27.01, 23.72; HRMS (ESI) calcd for C₁₈H₂₅N₄O₃(MH)⁺345.1921, found 345.1920.

2-amino-4-[3-(pyrimidin-2-ylcarbamoyl)-propyl]-imidazole-1-carboxylicacid tert-butyl ester (31). Tan solid (19 mg, 11%). ¹H NMR (400 MHz,DMSO-d₆) δ 10.51 (s, 1H), 8.63 (d, 2H, J=4.8 Hz), 7.15 (t, 1H, J=4.8Hz), 6.54 (s, 1H), 6.40 (s, 2H), 2.49 (t, 2H, J=7.2 Hz), 2.29 (t, 2H,J=7.2 Hz), 1.80 (quint., 2H, J=7.2 Hz), 1.53 (s, 9H); ¹³C NMR (75 MHz,DMSO-d₆) δ 171.34, 158.11, 157.63, 149.76, 148.85, 138.31, 116.37,105.81, 83.98, 35.75, 27.44, 27.05, 23.37; HRMS (ESI) calcd forC₁₆H₂₃N₆O₃ (MH)⁺347.1826, found 347.1827.

General EDC/HOBt procedure:2-amino-4-(3-carboxy-propyl)-imidazole-1-carboxylic acid tert-butylester 22 (100 mg, 0.371 mmol), 1-hydroxybenzotriazole (100 mg, 0.742mmol) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(142 mg, 0.742 mmol) were dissolved in anhydrous DMF (3 mL). Theappropriate amine coupling partner (1.48 mmol) was then added and thesolution was stirred at ambient temperature until completion was evidentby TLC analysis. The reaction was concentrated under reduced pressureand the residue partitioned between ethyl acetate (20 mL) and water (10mL). The organic layer was successively washed with water (3×10 mL), a10% aqueous solution of citric acid (2×10 mL), sat. NaHCO₃ (2×10 mL),and brine (10 mL) before being dried (Na₂SO₄) and evaporated to dryness.The crude product was purified via flash column chromatography (2-10%MeOH/CH₂Cl₂) to afford the target compound.

2-amino-4-(3-hexylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (24). Pale yellow solid (41 mg, 32%). ¹H NMR (300 MHz,DMSO-d₆) δ 7.73 (m, 1H), 6.50 (s, 1H), 6.39 (s, 2H), 2.99 (q, 2H, J=6.3Hz), 2.21 (t, 2H, J=7.5 Hz), 2.04 (t, 2H, J=7.2 Hz), 1.70 (m, 2H), 1.53(s, 9H), 1.31 (m, 3H), 1.23 (br s, 7H), 0.85 (t, 3H, J=5.1 Hz); ¹³C NMR(75 MHz, DMSO-d₆) δ 171.68, 149.93, 148.95, 138.37, 105.81, 84.04,38.35, 34.91, 31.01, 29.14, 27.52, 27.22, 26.10, 24.06, 22.09, 13.93;HRMS (ESI) calcd for C₁₈H₃₃N₄O₃ (MH)⁺353.2547, found 353.2549.

2-amino-4-(3-octylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (25). White solid (48 mg, 34%). ¹H NMR (300 MHz,DMSO-d₆) δ 7.73 (m, 1H), 6.50 (s, 1H), 6.38 (s, 2H), 2.99 (q, 2H, J=5.4Hz), 2.21 (t, 2H, J=7.5 Hz), 2.04 (t, 2H, J =7.2 Hz), 1.73 (m, 2H), 1.53(s, 9H), 1.36 (m, 4H), 1.23 (br s, 10H), 0.85 (t, 3H, J=5.1 Hz); ¹³C NMR(75 MHz, DMSO-d₆) δ 171.72, 149.95, 148.95, 138.43, 105.83, 84.06,38.35, 34.92, 31.26, 29.16, 28.71, 27.53, 27.22, 26.43, 24.10, 22.12,13.98; HRMS (ESI) calcd for C₂₀H₃₇N₄O₃ (MH)⁺381.2860, found 381.2861.

2-amino-4-(3-dodecylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester (27). White solid (44 mg, 28%). ¹H NMR (400 MHz,DMSO-d₆) δ 7.73 (t, 1H, J=5.6 Hz), 6.50 (s, 1H), 6.38 (s, 2H), 3.00 (q,2H, J=5.6 Hz), 2.21 (t, 2H, J=7.6 Hz), 2.04 (t, 2H, J=7.6 Hz), 1.71(quint., 2H, J=7.6 Hz), 1.53 (s, 9H), 1.36 (m, 2H), 1.23 (s, 18H), 0.85(t, 3H, J=6.0 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 171.58, 149.77, 148.88,138.42, 105.76, 83.98, 38.28, 34.90, 31.18, 29.06, 28.88, 28.58, 27.47,27.18, 26.29, 24.04, 21.96, 13.81, 13.27; HRMS (ESI) calcd forC₂₄H₄₅N₄O₃ (MH)⁺437.3486, found 437.3487.

4-(2-amino-1H-imidazol-4-yl)-N-isobutyl-butyramide hydrochloride (8). Asolution of2-amino-4-(3-isobutylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester 23 (76 mg, 0.234 mmol) in anhydrous dichloromethane (1mL) was cooled to 0° C. TFA (1 mL) was charged into the flask and thereaction stirred for 5 h. After that time the reaction was evaporated todryness and toluene (2 mL) was added. Again the mixture was concentratedand the process repeated. The resulting TFA salt was dissolved indichloromethane (1 mL) and 2M HCl in diethyl ether (0.50 mL) was addedfollowed by cold diethyl ether (8 mL). The precipitate was collected byfiltration and washed with diethyl ether (3 mL) to yield the targetcompound 8 (59 mg, 97%) as a tan solid. ¹H NMR (300 MHz, DMSO-d₆) δ12.14 (s, 1H), 11.70 (s, 1H), 7.89 (m, 1H), 7.34 (br s, 2H), 6.55 (s,1H), 2.84 (t, 2H, J=6.6 Hz), 2.38 (t, 2H, J=7.5 Hz), 2.10 (t, 2H, J=7.5Hz), 1.60-1.79 (m, 3H), 0.82 (d, 6H, J=6.3 Hz); ¹³C NMR (75 MHz,DMSO-d₆) δ 171.52, 146.78, 126.31, 108.68, 46.00, 34.41, 28.09, 23.94,23.64, 20.18; HRMS (ESI) calcd for C₁₁H₂₁N₄O (MH)⁺225.1709, found225.1711.

4-(2-amino-1H-imidazol-4-yl)-N-hexyl-butyramide hydrochloride (9). Usingthe same general procedure as used for the synthesis of 8,2-amino-4-(3-hexylcarbamoyl-propyl) -imidazole-1-carboxylic acidtert-butyl ester 24 (90 mg, 0.255 mmol) gave 9 (70 mg, 96%) as a paleyellow foam. ¹H NMR (300 MHz, DMSO-d₆) δ 11.96 (s, 1H), 11.54 (s, 1H),7.81 (m, 1H), 7.29 (br s, 2H), 6.56 (s, 1H), 3.01 (m, 2H), 2.40 (t, 2H,J=7.8 Hz), 2.07 (t, 2H, J=7.2 Hz), 1.73 (m, 2H), 1.23-1.36 (m, 8H) 0.85(m, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.35, 146.72, 126.37, 108.71,38.43, 34.41, 31.00, 29.12, 26.12, 23.87, 23.62, 22.09, 13.96; HRMS(ESI) calcd for C₁₃H₂₅N₄O (MH)⁺253.2022, found 253.2025.

4-(2-amino-1H-imidazol-4-yl)-N-octyl-butyramide hydrochloride (10).Using the same general procedure as used for the synthesis of 8,2-amino-4-(3-octylcarbamoyl-propyl) -imidazole-1-carboxylic acidtert-butyl ester 25 (50 mg, 0.131 mmol) gave 10 (39 mg, 93%) as a whitesolid. ¹H NMR (300 MHz, DMSO-d₆) δ 12.13 (s, 1H), 11.69 (s, 1H), 7.87(m, 1H), 7.33 (br s, 2H), 6.55 (s, 1H), 2.99 (q, 2H, J=6.3 Hz), 2.38 (t,2H, J=7.5 Hz), 2.07 (t, 2H, J=7.5 Hz), 1.73 (m, 2H), 1.35 (m, 2H), 1.23(m, 10H), 0.85 (t, 3H, J=6.3 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 171.30,146.80, 126.32, 108.57, 38.40, 34.39, 31.15, 29.06, 28.62, 28.56, 26.37,23.86, 23.57, 21.99, 13.85; HRMS (ESI) calcd for C₁₅H₂₉N₄O(MH)⁺281.2335, found 281.2339.

4-(2-amino-1H-imidazol-4-yl)-N-decyl-butyramide hydrochloride (11).Using the same general procedure as used for the synthesis of 8,2-amino-4-(3-decylcarbamoyl-propyl) -imidazole-1-carboxylic acidtert-butyl ester 26 (32 mg, 0.078 mmol) gave 11 (27 mg, 99%) as a whitesolid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.07 (s, 1H), 11.64 (s, 1H), 7.85(s, 1H), 7.32 (br s, 2H), 6.56 (s, 1H), 3.00 (q, 2H, J=6.4 Hz), 2.38 (t,2H, J=7.2 Hz), 2.07 (t, 2H, J=7.2 Hz), 1.73 (quint., 2H, J=7.2 Hz), 1.36(m, 2H), 1.23 (s, 14H), 0.85 (t, 3H, J=7.2 Hz); ¹³C NMR (100 MHz,DMSO-d₆) δ 171.33, 146.72, 126.36, 108.70, 38.42, 34.41, 31.32, 29.15,29.04, 28.99, 28.77, 28.73, 26.45, 23.89, 23.62, 22.12, 13.99; HRMS(ESI) calcd for C₁₇H₃₃N₄O (MH)⁺ 309.2648, found 309.2647.

4-(2-amino-1H-imidazol-4-yl)-N-dodecyl-butyramide hydrochloride (12).Using the same general procedure as used for the synthesis of 8,2-amino-4-(3-dodecylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester 27 (20 mg, 0.046 mmol) gave 12 (16 mg, 94%) as a whitesolid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.03 (s, 1H), 11.60 (s, 1H), 7.83(t, 1H, J=6.4 Hz), 7.31 (s, 2H), 6.56 (s, 1H), 3.00 (q, 2H, J=6.4 Hz),2.38 (t, 2H, J=7.2 Hz), 2.07 (t, 2H, J=7.2 Hz), 1.73 (quint., 2H J=7.2Hz), 1.36 (m, 2H), 1.23 (s, 18H), 0.85 (t, 2H, J=6.4 Hz); ¹³C NMR (75MHz, DMSO-d₆) δ 171.20, 146.66, 126.34, 108.59, 38.34, 34.32, 34.32,31.15, 29.02, 28.86, 28.6, 28.55, 26.31, 23.78, 23.52, 21.94, 13.79;HRMS (ESI) calcd for C₁₉H₃₇N₄O (MH)⁺ 337.2961, found 337.2964.

4-(2-amino-1H-imidazol-4-yl)-N-cyclopentyl-butyramide hydrochloride(13). Using the same general procedure as used for the synthesis of 8,2-amino-4-(3-cyclopentylcarbamoyl-propyl)-imidazole-1-carboxylic acidtert-butyl ester 28 (100 mg, 0.297 mmol) gave 13 (78 mg, 96%) as a paleyellow foam. ¹H NMR (400 MHz, DMSO-d₆) δ 11.99 (s, 1H), 11.57 (s, 1H),7.79 (d, 1H, J=7.2 Hz), 7.30 (s, 2H), 6.56 (s, 1H), 3.97 (m, 1H), 2.37(t, 2H, J=7.2 Hz), 2.05 (t, 2H, J=7.2 Hz), 1.69-1.79 (m, 4H), 1.59 (m,2H), 1.47 (m, 2H), 1.31 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.97,146.74, 126.38, 108.72, 50.11, 34.36, 32.31, 23.81, 23.45, 23.62; HRMS(ESI) calcd for C₁₂H₂₁N₄O (MH)⁺ 237.1709, found 237.1711.

4-(2-amino-1H-imidazol-4-yl)-1-morpholin-4-yl-butan-1-one hydrochloride(14). Using the same general procedure as used for the synthesis of 8,2-amino-4-(4-morpholin-4-yl-4-oxo-butyl)-imidazole-1-carboxylic acidteri-butyl ester 29 (44 mg, 0.133 mmol) gave 14 (25 mg, 70%) as a tansolid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.1 (s, 1H), 11.64 (s, 1H), 7.33 (s,2H), 6.58 (s, 1H), 3.54 (m, 4H), 3.42 (m, 4H), 2.43 (t, 2H, J=7.2 Hz),2.33 (t, 2H, J=7.2 Hz), 1.75 (quint., 2H, J=7.2 Hz); ¹³C NMR (75 MHz,DMSO-d₆) δ 170.27, 146.72, 126.39, 108.61, 66.04, 45.28, 31.05, 23.56,23.17; HRMS (ESI) calcd for C₁₁H₁₉N₄O₂ (MH)⁺ 239.1502, found 239.1503.

4-(2-amino-1H-imidazol-4-yl)-N-phenyl-butyramide hydrochloride (15).Using the same general procedure as used for the synthesis of 8,2-amino-4-(3-phenylcarbamoyl-propyl) -imidazole-1-carboxylic acidtert-butyl ester 30 (80 mg, 0.232 mmol) gave 15 (64 mg, 99%) as a tansolid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.03 (s, 1H), 11.60 (s, 1H), 9.98(s, 1H), 7.59 (d, 2H, J=8.0 Hz), 7.33 (br s, 2H), 7.28 (t, 2H, J=8.0Hz), 7.02 (t, 1H, J=7.6 Hz), 6.61 (s, 1H), 2.44 (m, 2H), 2.32 (t, 2H,J=6.8 Hz), 1.85 (m, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 170.65, 146.77,139.25, 137.24, 128.59, 126.40, 123.00, 119.13, 108.83, 35.33, 23.60,23.51; HRMS (ESI) calcd for C₁₃H₁₇N₄O (MH)⁺ 245.1396, found 245.1401.

4-(2-amino-1H-imidazol-4-yl)-N-pyrimidin-2-yl-butyramide hydrochloride(16). Using the same general procedure as used for the synthesis of 8,2-amino-4-[3-(pyrimidin-2-ylcarbamoyl) -propyl]-imidazole-1-carboxylicacid tert-butyl ester 31 (50 mg, 0.144 mmol) gave 16 (41 mg, 99%) as awhite solid. ¹H NMR (400 MHz, DMSO-d₆) δ 12.06 (s, 1H), 11.64 (s, 1H),10.59 (s, 1H), 8.64 (d, 2H, J=4.8 Hz), 7.34 (s, 2H), 7.17 (t, 1H, J=4.8Hz), 6.60 (s, 1H), 2.51 (m, 2H), 2.46 (t, 2H, J=7.2 Hz), 1.83 (quint.,2H, J=7.2 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 158.17, 157.54, 146.73,129.02, 126.33, 116.5, 108.71, 35.35, 23.48, 23.09; HRMS (ESI) calcd forC₁₁H₁₅N₆O (MH)⁺ 247.1301, found 247.1304.

EXAMPLE 5

Analysis of a library of oroidin derivatives. Two members of the oroidinfamily, bromoageliferin and oroidin, were documented to possessanti-biofouling properties by inhibiting biofilm development in themarine α-proteobacterium R. salexigens. A. Yamada et al., Bull. Chein.Soc. Jpn. 1997, 70, 3061. Herein we provide the results of astructure-activity relationship (SAR) analysis from the synthesis andbiological evaluation of a 50 compound oroidin library in the context ofanti-biofilm activity against the medically relevant gram-negativeγ-proteobacterium P. aeruginosa.

Marine natural products provide a diverse array of chemical structuresand are known to possess a plethora of biological activities. M. D.Lebar, et al., Nat. Prod. Rep. 2007, 24, 774. Most mernbers of theoroidin alkaloid family have nitrogen dense architectures that contain a2-aminoimidazole (2-AI) subunit. H. Hoffmann, T. Lindel,Synthesis-Stuttgart 2003, 1753. S. M. Weinreb, Nat. Prod. Rep. 2007, 24,931. These compounds are typically found in sponges of the familyAgelasidae and mainly serve as a chemical anti-feeding defense mechanismagainst predators. J. C. Braekman, et al., Biochem. Syst. Ecol. 1992,20, 417. Oroidin is believed to be one of the main building blocks inthe biosynthesis of other more complex family members includingpalau'amine and the stylissadines. A. Al Mourabit, P. Potier, Eur. J.Org. Chem. 2001, 237. M. Kock, et al., Angew. Chem., Int. Ed. 2007, 46,6586. In addition to being documented to interfere with the biofoulingprocess of R. salexigens, oroidin has also been observed to retardbacterial attachment and colonization in a limited number of studies. S.R. Kelly, et al., Aquat. Microb. Ecol. 2005, 40, 191. S. R. Kelly, etal., Aquat. Microb. Ecol. 2003, 31, 175.

A library of analogues was synthesized based upon the oroidin template.The structure-activity relationships (SAR) were then delineated withinthe context of anti-biofilm activity. Molecules based on oroidin wouldrequire a relatively short reaction sequence to access (2-6 steps) andcould be rapidly assembled from core scaffolds and screened for theiranti-biofilm properties.

Using this natural product as our base, a focused library wasconstructed by systematically varying three regions within the oroidintemplate (FIG. 15) to delineate what structural features of the moleculewere essential for biological activity. These areas were designated as:the tail group (Region A), the linker chain (Region B), and the headgroup (Region C). The tail group was varied as: absent, an N—H pyrrolederivative, or an N-methyl pyrrole derivative. The linker between thehead group and tail group was varied from two to four carbons and theeffect of chain unsaturation was also examined. The head groupsconsidered for analysis included 2-aminoimidazole,2-amino-4-oxoimidazole, imidazole, tryptoplian, 2-thioimidazolone, and2-aminothiazole (FIG. 16).

To examine each compound's ability to inhibit the formation ofPseudomonas aerziguiosa biofilms, PAO1 and PA14 were employed as thetarget bacterial strains using a crystal violet reporter assay. G. A.O'Toole, R. Kolter, Mol. Microbiol. 1998, 28, 449. All compounds wereinitially screened at 500 μM for anti-biofilm activity. IC₅₀ values werethen determined for compounds that displayed exceptional activity in thepreliminary screen followed by growth curve and colony count analysis toverify that the compounds were in fact true inhibitors of bacterialbiofilm formation and not acting as microbicides inducing cell deathbefore biofilm development had begun.

Region A SAR: Tail-group analogue synthesis and biological activity.Nearly all oroidin alkaloids are known to contain the pyrrolecarboxamide moiety with various degrees of bromination and this providedthe first structural element for investigation. H. Hoffmann, T. Lindel,Synthesis-Stuttgart 2003, 1753. Each analogue was prepared by aconvergent synthetic approach with amide bond formation between thescaffold 4-(3-aminopropyl)-2-aminoimidazole dihydrochloride 16 and theappropriate trichloroacetyl pyrrole derivative serving as the finalstep. Trichloroacetyl pyrroles are known to undergo smooth amide bondformation in the presence of an unprotected 2-aminoimidazole and areamong the most frequently used reagents in the total synthesis of manyoroidin relatives. V. B. Birman, X. T. Jiang, Org. Lett. 2004, 6, 2369.D. P. O'Malley, et al., J. Am. Chem. Soc. 2007, 129, 4762. The necessarytrichloroacetyl pyrroles were synthesized as outlined in Scheme 6. D. M.Bailey, R. E. Johnson, J. Med. Chem. 1973, 16, 1300. The correspondingN—H and N-methyl dibromo carboxylic acids 11 and 15 were also prepared.These simple compounds are frequently isolated in high concentrations inconjunction with the more complex oroidin alkaloids from the Agelasidaesponges and would serve as controls in the inhibition assay. A. E.Wright, et al., J. Nat. Prod. 1991, 54, 1684. A compiled activity listof all compounds synthesized and assayed at 500 μM for Region A SAR issummarized in Scheme 7.

SCHEME 7 Region A SAR synthesis and biological evaluation.

% Biofilm % Biofilm % Inhibition at Inhibition at Yield 500 μM vs PAO1500 μM vs PA14 17 R = X = Y = H 63 <10  16 18 R = H, X = H, Y = Br 74 7486 19 R = H, X = Y = Br 59 61 37 20 R = H, X = Y = Cl 65 37 48 21 R =CH₃, X = Y = H 58 25 — 22 R = CH₃, X = H, 56 88 83 Y = Br 23 R = CH₃, X= Y = Br 63 >95  >95 

The N—H pyrrole sub-class was the first group of analogues studied. Thedihydro derivatives of the natural products clathrodin, (J. J. Morales,A. D. Rodriguez, J. Nat. Prod. 1991, 54, 629) hymenidin, (J. Kobayashi,et al. Experientia 1986, 42, 1176) and oroidin represent the varioussuccessive degrees of N—H pyrrole bromination and were synthesized andscreened for their ability to inhibit the formation of P. aeruginosabiofilms (Scheme 7). As previously reported, scaffold 16 was relativelyinactive against both strains (20% inhibition against PAO1, 15% againstPA14). R. W. Huigens, et al., J. Am. Chem. Soc. 2007, 129, 6966.Dihydroclathrodin (DHC, 17) showed similar activity to the base scaffoldwith <10% inhibition of PAO1 and 16% inhibition of PA14 biofilmformation. Dihydrohymenidin (DHH, 18) showed a remarkable increase inactivity, inhibiting the formation of PAO1 and PA14 biofilms by 74% and86%, respectively. Addition of a second bromine atom at the 2-positionon the pyrrole ring yielded dihydrooroidin (DHO, 19). It washypothesized that activity would yet again increase but surprisingly DHOdisplayed a decrease in potency against both strains (PAO1inhibition=61%, PA14 inhibition=37%).

The requirement of a particular halogen identity on the pyrrole ring wasalso examined by replacing both bromine atoms with less stericallydemanding and less electronegative chlorine atoms (20). No known oroidinfamily members possess chlorine substituents on the pyrrole carboxamidesubunit yet some do contain chlorinated positions in other parts of themolecule. M. Kock, et al., Angew. Chem., Int. Ed. 2007, 46, 6586. Thisventure however proved unfruitful as no substantial benefit was gainedwith the dichloro derivative 20, inhibiting the formation of PAO1biofilms by 37% and PA14 biofilms by 48% at 500 μM.

Investigation into how introduction of a methyl substituent on thepyrrole nitrogen would affect activity was the next step in the SARprocess for this region. This decision was based upon the observationthat some naturally occurring members of the oroidin family (i.e.sventrin 24) contain an N-methylated pyrrole instead of the morecommonly seen N—H pyrrole moiety. M. Assmann, et al., J. Nat. Prod.2001, 64, 1593. The non-brominated derivative 21 showed a slightincrease in activity when compared to DHC, as it was observed that 21inhibited the formation of PAO1 biofilms by 25%. However, thisderivative was found to be inactive against PA14. Analogous to what wasobserved with the N—H pyrrole subset, N-methyl dihydrohymenidin 22showed a substantial increase in activity (88% and 83% inhibition forPAO1 and PA14, respectively). This time addition of a second bromine wasobserved to enhance activity as this compound, dihydrosventrin (DHS,23), inhibited the formation of both PAO1 and PA14 biofilms by >95% at500 μM.

All compounds that revealed >70% biofilm inhibition activity during thepreliminary 500 μM screen were selected for further biologicalcharacterization. Dose response curves were generated for each compoundto determine the analogue's IC₅₀ value against both Pseudomonas strains.These results are summarized in Table 4. Of the tail-modifiedderivatives, dihydrosventrin (DHS) was the most active with an IC₅₀ of51±9 μM against PAO1 and 111±8 μM against PA14. When the tail fragment15 was tested alone, it displayed no activity at 500 μM. This evidencefurther reinforces the fact that the two fragments must be covalentlylinked to elicit their anti-biofilm activity. Growth curves and colonycounts were also performed for both PAO1 and PA14 in the presence andabsence of DHS 23 at its respective IC₅₀ concentration. In each case, noreduction in bacterial density or viable colonies was observed, thusconfirming that our compounds were true inhibitors of biofilm formationand not eliciting their activity through a microbiocidal mechanism (notshown).

TABLE 4 IC₅₀ values for Region A analogues. Compound PAO1 IC₅₀(μM) PA14IC₅₀(μM) Dihydrohymenidin (18) 323 ± 30 266 ± 23 N-methyldihydrohymenidin (22) 348 ± 13 309 ± 16 Dihydrosventrin (23) 51 ± 9 111± 8 

The data gathered from this section of the SAR indicated a roughcorrelation between degree of bromination on the pyrrole ring andincreased anti-biofilm activity. It was also observed that compoundsbearing the N-methylated pyrrole had better potential as biofilminhibitors, illustrated by the remarkable difference in activity betweendihydrosventrin 23 and dihydrooroidin 19.

Region B SAR: Linker analogue synthesis and biological activity. Thedouble bond found in oroidin is proposed to have a profound impact onthe ability of the sponge to synthesize a number of more complexchemical skeletons (i.e. ageliferins, sceptrins) through dimerizationtype reactions. A. Al Mourabit, P. Potier, Eur. J. Org. Chem. 2001, 237.Discerning whether or not unsaturation was necessary for a biologicalresponse from an anti-biofilm standpoint would allow us to circumvent alow yielding extra synthetic step needed to install the double bondbetween the 3-4 positions in the dihydro scaffold 16. Oroidin 5 wasprepared as previously reported. A. Olofson, et al., J. Org. Chem. 1998,63, 1248. Sventrin 24 was synthesized using an identical syntheticapproach executed for oroidin with the exception of employing 14 in theamide bond formation step. Initial screens at 500 μM revealed that bothnatural products inhibited the formation of PAO1 and PA14 biofilms >95%.The activity of oroidin 5 (PAO1 IC₅₀=190±9 μM, PA14 IC₅₀=166±19 μM) wasexceptionally better than its dihydro congener, which was not evenconsidered a candidate for IC₅₀ value determination (vide supra). Incontrast, the IC₅₀ values of sventrin 24 (IC₅₀=75±5 μM PAO1, IC₅₀=115±3μM PA14) were very similar to those of its saturated counterpart (FIG.17). This seemed to indicate that as we begin to fine tune our scaffoldsto maximize anti-biofilm activity, unsaturation within the linker is notnecessary to elicit maximum biological activity. As carried out withDHS, follow tip growth curve and colony count analysis of PAO1 and PA14grown in the presence or absence of either natural product at theirrespective IC₅₀ concentrations did not induce microbial cell death.

Given that a fully saturated chain, when coupled to the4,5-dibromo-N-methylpyrrole subunit yielded a compound (DHS) with thehighest activity, we then elected to study the effect that linker lengthhad upon biological activity. Homologues of DHS that contained a2-methylene and a 4-methylene spacer between the 2-Al head and thepyrrole tail were envisioned. These compounds were quickly accessed asoutlined in Scheme 8. Briefly, commercially available1,4-diamino-2-butanone dihydrochloride 25 was condensed with cyanamideunder pH-controlled conditions to yield the 2-methylenie spacer 2-Al 26(T. Vitali, et al., Farmaco 1984, 39, 70), which was subsequentlycoupled to fragment 14 to deliver target 27. The 4-methylene spacer wasgenerated through Akabori reduction of lysine methyl ester 28 to producethe corresponding α-amino aldehyde, (Akabori, Ber. Deut. Chem. Ges.1933, 66, 151; G. C. Lancini, E. Lazzari, J. Heterocycl. Chem. 1966, 3,152) which, upon cyclization with cyanamide and ensuing amide bondformation, afforded the 2-Al 30.

Initial screens at 500 μM revealed that each compound, like the parentcompound DHS, inhibited the formation of PAO1 and PA14 biofilms by >95%.IC₅₀ values for both 27 and 30 did, however, indicate the subtle effectsthat alkyl linker length had upon activity, with both modificationsdecreasing activity in comparison to DHS. Increasing the alkyl chainlength to 4-methylene units elicited the smallest drop in activity (PAO1IC₅₀=150±17 μM, PA14 IC₅₀=126±17 μM), while the reduction in potency wasslightly more pronounced when decreasing the alkyl chain length to2-methylene units (PAO1 IC₅₀=165±23 μM, PA14 IC₅₀=224±22 μM). Colonycounts and growth curves performed with these homologues revealed nomicrobiocidal activity at their respective IC₅₀ values.

Screening of these various linker analogues quickly revealed twoimportant SAR features of the oroidin scaffold in terms of anti-biofilmactivity. First, the optimum chain length between the 2-AI head andpyrrole tail was three carbon units. Second, unsaturation was notnecessary to elicit a biological response, thus eliminating the need foran additional synthetic step which otherwise would have been needed foranalogue synthesis.

Region C SAR: Head-group analogue synthesis and biological activity.Given the ubiquitous nature of the 2-aminoimidazole group in oroidinalkaloids, a substantial effort was made to delineate the importance ofthe 2-AI head group. We first focused on determining the ramificationsof oxidizing the 2-AI ring at the 4-position. The natural productdispacamide (F. Cafieri, et al., Tet. Lett. 1996, 37, 3587) 31 and itsN-methyl congener 32 were synthesized and subsequently assayed forinhibition of PAO1 and PA14 biofilms (FIG. 18). Dispacamide was preparedas previously reported (A. Olofson, et al., J. Org. Chem. 1998, 63,1248) while dihydrosventrin 23 was also oxidized with molecular brominein DMSO to afford its requisite N-methyl analogue. Each compound showeda substantial reduction in activity with <20% PAO1 and PA14 biofilmformation inhibition at 500 μM.

The repercussions of atomic deletion or full head group replacementwithin Region C were investigated next. This was examined by replacementof the 2-AI group with a tryptophan residue or an imidazole grouplacking the 2-amino functionality. It was deemed unnecessary todelineate a synthesis for a 3-carbon linker of trytophan and imidazolewhen their 2-carbon homologues were commercially available and could bedirectly compared to the corresponding 2-AI derivative with a2-methylene unit linker which had already been characterized. Tryptaminehydrochloride or histamine dihydrochloride were coupled to all of thedifferent trichloroacetyl pyrroles discussed in the Region A SAR portionof this report and assayed for biofilm inhibition activity (Scheme 9).

SCHEME 9 Region C SAR synthesis and biological evaluation.

R = H, CH₃ X, Y = H, Cl, Br No actvity for the inhibition of either PAO1or PA14 bioflims

% Biofilm % Biofilm % Inhibition at Inhibition at Yield 500 μM vs PAO1500 μM vs PA14 34 R = X = Y = H 54 42 31 35 R = H, X = H, Y = Br 40 4643 36 R = H, X = Y = Br 35 73 69 37 R = H, X = Y = Cl 62 33 38 38 R =CH₃, X = Y = H 55 29 34 39 R = CH₃, X = H, 62 37 21 Y = Br 40 R = CH₃, X= Y = Br 60 73 82

Replacement of the 2-AI subunit with a tryptophan abolished all activityat 500 μM, no matter what pyrrole derivative was appended to the tail.Removal of the 2-amino group was not as deleterious, as each compound weinitially assayed at 500 μM showed varying degrees of anti-biofilmactivity against both PAO1 and PA14. IC₅₀ value determination ofanalogue 40 (IC₅₀=277±35 μM PAO1, IC₅₀=203±25 μM PA14) for comparison toits 2-Al 2-methylene spacer homologue 27 (PAO1 IC₅₀=165±23 μM, PA14IC₅₀=224±22 μM), revealed that in comparison to 27, a substantial dropin activity against PAO1 is noted along with slightly better activityagainst PA14 (FIG. 19). Subsequent growth curves and colony countsindicated that 40 was not inhibiting biofilm development throughmicrobiocidal activity.

Finally, we inquired how single atom changes within the 2-AI subunitwould affect anti-biofilm activity. To this end, we elected tosynthesize the 2-thioimidazolone and 2-aminothiazole (2-AT) scaffoldsfor SAR study. Condensation of an α-amino carbonyl compound with anisocyanate is well known, (A. C. B. Sosa, et al., Org. Lett. 2000, 2,3443) and provided the basis for the synthesis of the 2-thioimidazolonescaffold 42. Similar to the known route to access 2-AI scaffold 16,Akabori reduction of ornithine methyl ester followed immediately bycyclization with KSCN under pH controlled conditions afforded the 2-thioimidazolone 42. Acylation of the terminal amine was accomplishedwith conditions adopted from the Region A SAR study to afford 43-49 inmodest yields (Scheme 10). All derivatives in this subset were able toinhibit biofilm formation throughout a range of values at 500 μM.

SCHEME 10 Region C SAR synthesis and biological evaluation.

% Biofilm % Biofilm % Inhibition at Inhibition at Yield 500 μM vs PAO1500 μM vs PA14 43 R = X = Y = H 53 39 33 44 R = H, X = H, Y = Br 52 2814 45 R = H, X = Y = Br 41 61 56 46 R = H, X = Y = Cl 65 41 38 47 R =CH₃, X = Y = H 57 27 36 48 R = CH₃, X = H, 54 41 57 Y = Br 49 R = CH₃, X= Y = Br 71 46 38

2-AT's are known to possess biological activity and thus were deemed alogical choice for head group study. J. C. Eriks, et al., J. Med. Chem.1992, 35, 3239. J. L. Kane, et al., Bioorg. Med. Chem. Lett. 2003, 13,4463. To affect the synthesis of the 2-AT scaffold, a new synthetic planwas necessary to selectively install a sulphur atom at the 1-position inthe ring (Scheme 11). Synthesis commenced with acyl chloride formationof the known 4-phthalimidobutanoic acid 50. W. J. Kruper, et al., J.Org. Chem. 1993, 58, 3869. This was followed by diazomethanehomologation and concomitant quench with concentrated HBr, whichafforded the α-bromoketone. Cyclization of the α-bromoketone withthiourea under neutral conditions cleanly and regioselectively installedthe 2-AT ring (51). J. C. Eriks, et al., J. Med. Chem. 1992, 35, 3239.A. Hantzsch, V. Traumann, Berichte 1888, 21, 938. Deprotection of thephthlamide protecting group was accomplished with hydrazine in methanolto deliver the 2-AT scaffold. Again, acylation of the terminal amine wasaccomplished as previously outlined to afford the final targetanalogues. The 2-AT sub-library was completely inactive at 500 μM asseen with the tryptophan derivatives.

These single atom replacements concluded the SAR study of Region C.Oxidation of the 2-AI ring in DHS proved detrimental by eliminatingnearly all biological activity. In addition, an imidazole-based motifremained important in the ability of these compounds to inhibit theformation of Pseudomonas biofilms despite trading out the 2-AI subunitfor a variety of functionally unique moieties.

In conclusion, through the generation of a 50-compound library, numeroustrends become apparent when the SAR data is reviewed in the context ofanti-biofilm activity. First, a 3-methylene linker between the 2-AI headand pyrrole tail elicits maximum biological activity. Second,unsaturation within the linker does not appear to be necessary toaugment biological response once the other regions of the oroidintemplate are fine-tuned for maximum activity. Third, an imidazole or2-AI head is necessary to maintain activity. Fourth, derivatives thatcontain di-bromonated N-methyl pyrroles have the tendency to be the mostpotent analogues within their respective sub-libraries. These trendsculminated in the identification of a lead candidate, DHS 23, as a verypotent and non-toxic inhibitor of Pseudomonas biofilm formation.

Experimental. Stock solutions of all compounds assayed for biologicalactivity were prepared in DMSO and stored at room temperature. Theamount of DMSO used in both inhibition and dispersion screens did notexceed 1% (by volume). Preliminary screens at 500 μM were performed induplicate. IC₅₀ dose response assays were preformed in triplicate ormore. P. aeruginosa strains PAO1 and PA14 were graciously supplied bythe Wozniak group at Wake Forest University School of Medicine.

General Static Inhibition Assay Protocol for Pseudomonas aeruginosa. Anovernight culture of the wild type strain was subculture at an OD₆₀₀ of0.10 into LBNS along with a predetermined concentration of the smallmolecule to be tested for biofilm inhibition. Samples were thenaliquoted (100 μL) into the wells of a 96-well PVC microtiter plate. Themicrotiter dishes were covered and sealed before incubation understationary conditions at 37° C. for 24 hours. After that time, themedium was discarded and the plates thoroughly washed with water. Thewells were then inoculated with a 0.1% aqueous solution of crystalviolet (100 μL) and allowed to stand at ambient temperature for 30minutes. Following another thorough washing with water the remainingstain was solubilized with 200 μL of 95% ethanol. Biofilm inhibition wasquantitated by measuring the OD₅₄₀ for each well by transferring 125 μLof the ethanol solution into a fresh polystyrene microtiter dish foranalysis.

All reagents including anhydrous solvents used for the chemicalsynthesis of the library were purchased from commercially availablesources and used without further purification unless otherwise noted.All reactions were run under either a nitrogen or argon atmosphere.Flash silica gel chromatography was performed with 60 Å mesh standardgrade silica gel from Sorbtech. ¹H and ¹³C NMR spectra were obtainedusing Varian 300 MHz or 400 MHz spectrometers. NMR solvents werepurchased from Cambridge Isotope Labs and used as is. Chemical shiftsare given in parts per million relative to DMSO-d₆ (δ 2.50), CD₃OD (δ3.31) and CDCl₃ (δ 7.27) for proton spectra and relative to DMSO-d₆ (δ39.51), CD₃OD (δ 49.00) and CDCl₃ (δ 77.21) for carbon spectra with aninternal TMS standard. High-resolution mass spectra were obtained at theNorth Carolina State Mass Spectrometry Laboratory for Biotechnology. FABexperiments were carried with a JOEL HX110HF mass spectrometer while ESIexperiments were carried out on Agilent LC-TOF mass spectrometer.

1-(4-bromo-1H-pyrrol-2-yl)-2,2,2-trichloro-ethanone (7).2-trichloroacetyl pyrrole 6 (5.00 g, 23.3 mmol) was dissolved inanhydrous chloroform (20 mL). The solution was cooled to −10° C. beforethe drop-wise addition of bromine (1.20 mL, 23.3 mmol) to the flask.Once addition was complete the reaction was allowed to warm to roomtemperature on its own accord while stirring for an additional 30minutes. The reaction was poured into water (40 mL) and extracted withchloroform (3×20 mL). The combined organic layers were washed with sat.NaHCO₃ (2×30 mL), brine (1×20 mL), dried over anhydrous sodium sulfate,filtered, and evaporated to dryness. Purification of the residue bycolumn chromatography (Hexanes/Diethyl Ether 95:5) yielded the titlecompound 7 (6.37 g, 93%) as an off-white solid: ¹H NMR (300 MHz,DMSO-d₆) δ 12.86 (s, 1H), 7.54 (s, 1H), 7.32 (s, 1H); ¹³C NMR (100 MHz,DMSO-d₆) δ 171,67, 129.06, 122.01, 121.54, 97.60, 94.56; HRMS (FAB)calcd for C₆H₃BrCl₃NO (M⁺) 288.8464, found 288.8479.

1-(4-bromo-1-methyl-pyrrol-2-yl)-2,2,2-trichloro-ethanone (13). Usingthe same general procedure as used for the synthesis of1-(4-bromo-1H-pyrrol-2 -yl)-2,2,2-trichloro-ethanone 7, 5.00 g of2-trichloroacetyl-1-methyl pyrrole afforded 5.46 g (81%) of the titlecompound 13 as a white solid: ¹H NMR (300 MHz, DMSO-d₆) δ 7.66 (d, 1H,J=1.2 Hz), 7.42 (d, 1H, J=1.8 Hz), 3.91 (s, 3 H); ¹³C NMR (100 MHz,DMSO-d₆) δ 171.48, 134.40, 123.62, 121.19, 95.36, 95.12; HRMS (FAB)calcd for C₇H₆BrCl₃NO (MH⁺) 303.8698, found 303.8678.

2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)-ethanone (8).2-trichloroacetyl pyrrole 6 (5.00 g, 23.3 mmol) was dissolved inanhydrous chloroform (20 mL). The solution was cooled to −10° C. beforethe drop-wise addition of bromine (2.64 mL, 51.3 mmol) to the reaction.Once addition was complete the reaction was allowed to warm to roomtemperature on its own accord while stifling for an additional 30minutes. The reaction was poured into water (40 mL) and extracted withchloroform (3×20 mL). The combined organic layers were washed with sat.NaHCO₃ (2×30 mL), brine (1×20 mL), and dried over anhydrous sodiumsulfate. Filtration and evaporation afforded the crude product which wasrecrystallized from hexanes to deliver 7.93 g (91%) of the titlecompound 8 as an off-white solid: ¹H NMR (300 MHz, DMSO-d₆) δ 13.75 (s,1H), 7.40 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.94, 123.30, 122.45,114.62, 100.88, 94.08; HRMS (FAB) calcd for C₆H₂Br₂Cl₃NO (M⁺) 366.7569,found 366.7556.

2,2,2-trichloro-1-(4,5-dibromo-1-methyl-pyrrol-2-yl)-ethanone (14).Using the same general procedure as used for the synthesis of2,2,2-trichloro-1-(4,5 -dibromo-1H-pyrrol-2-yl) -ethanone 8, 5.00 g of2-trichloroacetyl-1-methyl pyrrole 12 gave 8.14 g (96%) of the titlecompound 14 as white needles. ¹H NMR (300 MHz, DMSO-d₆) δ 7.60 (s, 1H),3.96 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.86, 123.81, 122.68,120.58, 99.58, 94.89, 37.60; HRMS (FAB) calcd for C₇H₄Br—Cl₃NO (M⁺)380.7725, found 380.7744.

2,2,2-trichloro-1-(4,5-dichloro-1H-pyrrol-2-yl)-ethanone (9).2-trichloroacetyl pyrrole 6 (5.00 g, 23.5 mmol) was dissolved inanhydrous chloroform (10 mL) and the reaction flask was covered inaluminum foil to exclude light. Sulfuryl chloride (4.20 mL, 51.8 mmol),was then charged in the flask and the reaction was refluxed for 16 hbefore being cooled to room temperature and poured into cold water (100mL). The aqueous layer was removed and washed with dichloromethane (2×25mL). The combined organic layers were then washed with sat. NaHCO₃ (3×35mL), dried over anhydrous sodium sulfate, filtered, and concentrated invacuo. The residue was purified by column chromatography(Hexanes/Diethyl Ether 95:5) to afford 4.61 g (70%) of the desiredcompound 9 as a white solid: ¹H NMR (300 MHz, DMSO-d₆) δ 13.84 (s, 1H),7.41 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.2, 123.6, 119.9, 119.7,110.8, 94.8; HRMS (FAB) calcd for C₆H₂Cl₅NO (M⁺) 278.8579, found278.8573.

4,5-dibromo-1H-pyrrole-2-carboxylic acid (11). Pyrrole-2 -carboxylicacid 10 (1.00 g, 9.00 mmol), was dissolved in anhydrous chloroform (10mL) and glacial HOAc (2 mL). To the resulting cloudy solution was slowlyadded bromine (0.971 mL, 18.9 mmol) at room temperature and onceaddition was complete the reaction was heated to 50° C. for 5 h. Aftercooling to ambient temperature the reaction was partitioned betweenwater (30 mL) and chloroform (40 mL). The organic layer was rinsed withwater (2×30 mL) and 10% K₂CO₃ (40 mL). The K₂CO₃ extract was then washedwith chloroform (2×20 mL) and acidified to pH=3 with an aqueous solutionof 4N HCl. The precipitate was collected by vacuum filtration and thefilter cake rinsed with water (15 mL) to afford the target compound 11(2.10 g, 87%) as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 12.80 (bs,1H), 6.82 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.43, 125.37, 116.73,106.50, 98.70; HRMS (FAB) calcd for C₅H₃Br₂NO (M⁺) 266.8531, found266.8525.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid methyl ester. 2,2,2-trichloro-1-(4,5-dibromo-1-methyl-1H-pyrrol-2-yl)-ethanone 14 (1.00 g,2.60 mmol), anhydrous potassium carbonate (0.719 g, 5.20 mmol), andanhydrous methanol (20 mL) were charged into a reaction flask. Theresulting suspension was stirred for 16 h at room temperature upon whichthe reaction was quenched with water (10 mL). The methanol was removedunder reduced pressure and the residue partitioned between ethyl acetate(100 mL) and water (20 mL). The organic layer was subsequently washedwith sat. NaHCO₃ (2×30 mL), brine (2×20 mL), dried over anhydrous sodiumsulfate, and filtered. Evaporation of the filtrate yielded the titlecompound (0.710 g, 92%) as a white solid: 1H NMR (300 MHz, DMSO-d₆) δ7.05 (s, 1H), 3.90 (s, 3H), 3.76 (s, 3H); ¹³C NMR (75 MHz, DMSO-d₆) δ159.38, 123.62, 118.51, 113.93, 98.06, 51.58, 35.78; HRMS (FAB) calcdfor C₇H₇Br₂NO₂ (M⁺) 294.8844, found 294.8861.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid (15). 4,5-dibromo-1-methyl-1H-pyrrole-2-carboxylic acid methyl ester (0.675 g,2.27 mmol), lithium hydroxide (0.436 g, 18.19 mmol), methanol (12 mL),tetrahydrofuran (4 mL), and water (4 mL) were stirred for 16 h atambient temperature. The pH was then adjusted to 7.0 with an aqueoussolution of 4N HCl. The organics were removed by rotary evaporation andthe resulting residue diluted with water (15 mL). Acidification of theaqueous layer to pH=3 with 4N HCl afforded a white solid which wascollected by vacuum filtration. The filter cake was rinsed with water(10 mL) to give the title compound 15 (0.601 g, 94%) as a white solid:¹H NMR (400 MHz, DMSO-d₆) δ 12.83 (s, 1H), 7.00 (s, 1H), 3.90 (s, 3H);¹³C NMR (100 MHz, DMSO-d₆) δ 160.51, 124.74, 118.41, 113.07, 97.72,35.64; HRMS (FAB) calcd for C₆H₅Br₂NO₂ (M⁺) 280.8687, found 280.8676.

4-(3-amino-propyl)-1H-imidazol-2-ylamine dihydrochloride (16). Preparedas previously reported.^([37]) ¹H NMR (300 MHz, DMSO-d₆) δ 12.04 (br s,1H), 8.25 (br s, 2H), 7.41 (s, 2H), 6.65 (s, 1H), 2.75 (t, 2H, J=7.2),2.52 (m, 2H), 1.85 (tt, 2H, J=7.5, 14.7 Hz); ¹³C NMR (100 MHz, DMSO-d₆)δ 146.9, 125.4, 108.9, 37.7, 25.5, 21.1; HRMS (FAB) calcd for C₆H₁₂N₃S(MH⁺) 158.0752, found 158.0743.

1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride (17).4-(3-amino-propyl)-1H-imidazol-2-ylamine dihydrochloride 16 (0.100 g,0.458 mmol), 2-trichloroacetyl pyrrole 6 (0.103 g, 0.488 mmol), andanhydrous sodium carbonate (0.172 g, 1.63 mmol), were dissolved inanhydrous N,N-dimethylformamide (5 mL). The reaction was stirred atambient temperature for 16 h. Evaporation of the reaction under reducedpressure and purification of the residue by column chromatography(CH₂Cl₂/MeOH sat. NH₃ 85:15) afforded the desired compound in its freebase form. Addition of a single drop of concentrated hydrochloric acidto a methanol solution (8 mL) and evaporation under reduced pressureyielded 0.078 g (63%) of the title compound 17 as a white solid: ¹H NMR(300 MHz, DMSO-d₆) δ 12.07 (s, 1H), 11.59 (s, 1H), 11.45 (s, 1H), 8.14(t, 1H, J=5.1 Hz), 7.30 (s, 2H), 6.81 (m, 2H), 6.74 (s, 1H), 6.59 (s,1H), 6.04 (s, 1H), 3.19 (dt, 2H, J=6.6, 12.6 Hz), 2.42 (t, 2H, J=6.9Hz), (tt, 2H, J=6.6, 13.8 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.74,146.76, 126.43, 126.40, 121.13, 110.28, 108.65, 108.50, 37.59, 28.14,21.60; HRMS (ESI) calcd for C₁₁H₁₆N₅O (MH⁺) 234.1349, found 234.1354.

4-bromo-1H-pyrrole-2-carboxylic acid[3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride (18). Usingthe same general procedure as used for the synthesis of1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride 17, 0.132 g of4-(3-amino-propyl)-1H-imidazol-2-ylamine dihydrochloride 16 gave thetarget compound 18 (0.159 g, 74%) as an off-white solid: ¹H NMR (300MHz, DMSO-d₆) δ 12.20 (s, 1H), 11.85 (s, 1H), 11.57 (s, 1H), 8.23 (m,1H), 7.31 (s, 2H), 6.97 (d, 1H, J=1.5 Hz), 6.86 (d, 1H, J=1.5 Hz), 6.61(s, 1H), 3.21 (m, 2H), 2.44 (t, 2H, J=7.2 Hz), 1.73 (m, 2H); ¹³C NMR (75MHz, DMSO-d₆) δ 159.63, 146.68, 126.99, 126.40, 121.06, 111.61, 108.70,94.90, 37.67, 27.91, 21.56; HRMS (FAB) calcd for C₁₁H₁₅BrN₅O (MH⁺)312.0460, found 312.0475.

4,5-dibromo-1H-pyrrole-2-carboxylic acid[3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride (19). Usingthe same general procedure as used for the synthesis of1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amidehydrochloride 17, 0.100 g of 4-(3-amino-propyl)-1H-imidazol-2 -ylaminedihydrochloride 16 afforded 0.117 g (59%) of the title compound 19 as anoff-white solid: ¹H NMR (300 MHz, DMSO-d₆) δ 8.33 (t, 1H, J=5.4 Hz),7.07 (s, 2H), 6.95 (s, 1H), 6.56 (s, 1H), 3.22 (dt, 2H, J =6.0, 12.3Hz), 2.43 (t, 2H, J=7.2 Hz), 1.73 (tt, J=6.9, 13.8 Hz); ¹³C NMR (100MHz, DMSO-d₆) δ 158.92, 146.87, 128.31, 126.75, 112.88, 108.82, 104.33,97.76, 37.74, 27.89, 21.74; HRMS (FAB) calcd for C₁₁H₁₄Br₂N₅O (MH⁺)389.9565, found 389.9570.

4,5-dichloro-1H-pyrrole-2-carboxylic acid[3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride (20). Usingthe same general procedure as used for the synthesis of1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amidehydrochloride 17, 0.200 g of 4-(3-amino-propyl)-1H-imidazol-2 -ylaminedihydrochloride 16 afforded 0.204 g (65%) of the title compound 20 as awhite solid: ¹H NMR (300 MHz, DMSO-d₆) δ 8.32 (t, 1H, J=4.8 Hz), 6.91(s, 2H), 6.53 (s, 1H), 3.21 (dt, 2H, J=6.6, 12.6 Hz), 2.42 (t, 2H, J=7.2Hz), 1.72 (tt, 2H, J=7.5, 14.1 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 159.15,147.11, 127.31, 125.14, 114.75, 109.82, 108.99, 107.77, 37.83, 28.03,22.04; HRMS (ESI) calcd for C₁₁H₁₄C₁₂N₅O (MH⁺) 302.0569, found 302.0569.

1-methyl-pyrrole-2-carboxylic acid[3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride (21). Usingthe same general procedure as used for the synthesis of1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amidehydrochloride 17, 0.300 g of 4-(3-amino-propyl)-1H-imidazol-2-ylaminedihydrochloride 16 delivered 0.229 g (58%) of the target compound 21 asa pale yellow solid: ¹H NMR (300 MHz, DMSO-d₆) δ 8.03 (t, 1H, J=5.1 Hz),6.86 (m, 1H), 6.75 (m, 1H), 6.32 (s, 1H), 5.98 (m, 1H), 5.86 (br s, 2H),3.82 (s, 3H), 3.17 (dt, 2H, J=6.3, 13.2 Hz), 2.36 (t, 2H, J=7.2 Hz),(tt, 2H, J=7.2, 14.1 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 161.39, 148.11,129.74, 127.47, 125.72, 112.09, 109.63, 106.53, 38.00, 36.15, 28.65,23.30; HRMS (ESI) calcd for C₁₂H₁₈N₅O (MH⁺) 248.1506, found 248.1514.

4-bromo-1-methyl-pyrrole-2-carboxylic acid[3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride (22). Usingthe same general procedure as used for the synthesis of1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amidehydrochloride 17, 0.150 g of 4-(3-amino-propyl)-1H-imidazol-2 -ylaminedihydrochloride 16 afforded 0.142 g (56%) of the desired compound 22 asa pale yellow solid: ¹H NMR (300 MHz, DMSO-d₆) δ 8.17 (t, 1H, J=5.7 Hz),7.08 (d, 1H, J=1.2 Hz), 6.91 (s, 2H), 6.85 (d, 1H, J=1.5 Hz), 6.52 (s,1H), 3.80 (s, 3H), 3.17 (dt, 2H, J=6.3, 12.9 Hz), 2.41 (t, 2H, J=7.2Hz), 1.71 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.29, 147.23, 127.39,126.87, 126.43, 113.60, 108.94, 92.89, 37.75, 36.33, 28.02, 22.08; HRMS(ESI) calcd for C₁₂H₁₇BrN₅O (MH⁺) 326.0610, found 326.0613.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid[3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride (23). Usingthe same general procedure as used for the synthesis of1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amidehydrochloride 17, 0.200 g of 4-(3-amino-propyl)-1H-imidazol-2 -ylaminedihydrochloride 16 gave 0.258 g (63%) of the title compound 23 as awhite solid: ¹H NMR (300 MHz, DMSO-d₆) δ 12.06 (s, 1H), 11.59 (s, 1H),8.31 (t, 1H, J=5.4 Hz), 7.32 (s, 2H), 7.03 (s, 1H), 6.60 (s, 1H), 3.87(s, 3H), 3.18 (dt, 2H, J=6.3, 12.3 Hz), 2.45 (t, 2H, J=7.8 Hz), 1.73 (m,2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 159.77, 147.30, 127.99, 127.77, 114.00,110.43, 109.06, 96.86; 37.94, 35.38, 27.96, 22.28; HRMS (FAB) calcd forC₁₂H₁₆Br₂N₅O (MH⁺) 403.9722. found 403.9728.

oroidin hydrochloride (5). Prepared as previously reported.^([37]) ¹HNMR (300 MHz, DMSO-d₆) δ 12.78 (s, 1H), 12.54 (s, 1H), 11.89 (s, 1H),8.55 (t, 1H, J=6.0 Hz), 7.47 (s, 2H), 6.99 (d, 1H, J=3.0 Hz), 6.90 (s,1H), 6.17 (m, 2H), 3.95 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 158.73,147.46, 127.99, 126.85, 124.84, 116.15, 112.81, 111.15, 104.74, 97.91,39.83; HRMS (FAB) calcd for C₁₁H₁₂Br₂N₅O (MH⁺) 387.9409, found 387.9402.

sventrin hydrochloride (24). Using the same general procedure as usedfor the synthesis of 1H-pyrrole-2-carboxylic acid[3-(2-amino-1H-imidazol-4-yl)-propyl]-amide hydrochloride 17, 0.050 g of4-(3-amino-propenyl)-1H-imidazol-2 -ylamine dihydrochloride afforded0.062 g (61%) of sventrin hydrochloride 24 as a pale yellow solid: ¹HNMR (400 MHz, DMSO-d₆) δ 8.49 (t, 1H, J=5.6 Hz), 7.06 (s, 1H), 6.77 (s,2H), 6.75 (s, 1H), 6.19 (d, 1H, J=15.6 Hz), 6.02 (dt, 1H, J=5.6, 11.2Hz), 3.94 (m, 2H), 3.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 159.57,147.44, 127.59, 126.75, 124.85, 116.25, 114.17, 111.20, 110.88, 96.98,35.45; HRMS (ESI) calcd for C₁₂H₁₄Br₂N₅O (MH⁺) 401.9560, found 401.9560.

4-(2-amino-ethyl)-1H-imidazol-2-ylamine dihydrochloride (26).1,4-diamino-2-butanone dihydrochloride 25 (0.300 g, 1.71 mmol) andcyanamide (0.753 g, 17.9 mmol) were dissolved in water (10 mL). The pHof the solution was adjusted to pH=4.3 before heating the reaction at95° C. for 3.5 h while open to the atmosphere. After cooling to ambienttemperature ethanol (10 mL) was added to the flask and the solution wasevaporated to dryness. Purification of the residue by columnchromatography (MeOH sat. with NH₃/CH₂Cl₂ 90:10) yielded the product asits corresponding free base. Addition of methanol (10 mL) andconcentrated hydrochloric acid followed by evaporation in vacuo affordedthe target compound 26 (0.211 g, 62%) as a yellow solid: ¹H NMR (300MHz, DMSO-d₆) δ 6.21 (s, 1H), 5.14 (br s, 2H), 2.81 (m, 2H), 2.47 (m,2H); HRMS (ESI) calcd for C₅H₁₁N₄ (MH⁺) 127.0978, found 127.0977.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid[2-(2-amino-1H-imidazol-4-yl)-ethyl]-amide hydrochloride (27). Using thesame general procedure as used for the synthesis of1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amidehydrochloride 17, 0.150 g of 4-(2-amino-ethyl)-1H-imidazol-2 -ylaminedihydrochloride 26 afforded 0.206 g (64%) of the title compound 27 as anoff-white solid: ¹H NMR (300 MHz, DMSO-d₆) δ 8.24 (t, 1H, J=5.1 Hz),6.95 (s, 1H), 6.20 (s, 1H), 5.02 (s, 2H), 3.87 (s, 3H), 3.31 (m, 2H);¹³C NMR (75 MHz, DMSO-d₆) δ 159.54, 149.20, 128.14, 113.86, 113.69,110.33, 99.14, 96.83, 35.33, 27.45; HRMS (ESI) calcd for C₁₁H₁₄Br₂N₅O(MH⁺) 389.9559, found 389.9574.

4-(4-amino-butyl)-1H-imidazol-2-ylamine dihydrochloride (29). Using thesame general procedure as used for the synthesis of4-(3-amino-propyl)-1H-imidazol -2-ylamine dihydrochloride 16, 12.5 g oflysine methyl ester dihydrochloride 28 afforded 2.25 g (18%) of thetarget compound 29 as a yellow solid: ¹H NMR (300 MHz, DMSO-d₆) δ 6.09(s, 1H), 4.96 (s, 2H), 2.56 (t, 2H, J=6.3 Hz), 2.27 (t, 2H, J=6.9 Hz),1.35-1.51 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 149.11, 132.29, 110.59,40.75, 31.41, 26.62, 26.12; HRMS (ESI) calcd for C₇H₁₅N₄ (MH⁺) 155.1291,found 155.1293.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid[4-(2-amino-1H-imidazol-4-yl)-butyl]-amide hydrochloride (30). Using thesame general procedure as used for the synthesis of1H-pyrrole-2-carboxylic acid [3-(2-amino-1H-imidazol-4-yl)-propyl]-amidehydrochloride 17, 0.200 g of 4-(4-amino-butyl)-1H-imidazol-2 -ylaminedihydrochloride 29 delivered 0.216 g (54%) of the target compound 30 asa pale yellow solid: ¹H NMR (300 MHz, DMSO-d₆) δ 8.20 (t, 1H, J=5.1 Hz),6.99 (s, 1H), 6.35 (s, 1H), 6.29 (bs, 2H), 3.86 (s, 3H), 3.17 (m, 2H),2.35 (m, 2H), 1.49 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 159.66, 147.48,128.39, 128.06, 113.86, 110.37, 109.17, 96.85, 38.23, 35.35, 28.51,25.48, 24.53; HRMS (ESI) calcd for C₁₃H₁₈Br₂N₅O (MH⁺) 417.9873, found417.9870.

2-amino-5-(3-amino-propylidene)-1,5-dihydro-imidazol-4-onedihydrochloride. 4-(3-amino-propyl)-1H-imidazol-2-ylamine 16 (0.200 g,0.930 mmol) was dissolved in anhydrous dimethyl sulfoxide (6 mL).Bromine (0.047 mL, 0.930 mmol) was added drop-wise and the solution wasstirred at room temperature for 1 h. Diethyl ether (7 mL) was added andthe organics were then decanted. The residue was purified by columnchromatography (MeOH sat. with NH₃) to yield the desired product as itsfree base. Addition of concentrated hydrochloric acid to a methanolsolution (8 mL) of the free base followed by evaporation under reducedpressure afforded the target compound ((Z)-isomer exclusively) (0.141 g,67%) as a tan solid: ¹H NMR (300 MHz, DMSO-d₆) δ 12.10 (br s, 1H), 9.20(br s, 2H), 8.18 (br s, 2H), 5.92 (t, 1H, J=7.8 Hz), 2.96 (m, 2H), 2.66(m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 164.13, 156.63, 130.88, 113.70,37.57, 24.94; HRMS (FAB) calcd for C₆H₁₀N₄O (MH⁺) 155.0933, found155.0943.

dispacamide hydrochloride (31). Using the same general procedure as usedfor 2 -amino-5-(3-amino-propylidene)-1,5-dihydro-imidazol-4-onedihydrochloride, 0.185 g of dihydrooroidin hydrochloride 19 gave 0.120 g(63%) of dispacamide hydrochloride (8:1 Z/E isomer) 31 as a tan solid:¹H NMR (300 MHz, CD₃OD) (Z isomer) δ 6.79 (s, 1H), 6.14 (t, 1H, J=7.8Hz), 3.46 (t, 2H, J=6.9 Hz), 2.58 (dt, 2H, J=6.9, 14.7 Hz); ¹³C NMR (75MHz, DMSO-d₆) δ 164.32, 162.06, 157.58, 130.90, 128.75, 119.09, 114.51,106.47, 100.15, 39.11, 28.81; HRMS (ESI) calcd for C₁₁H₁₂Br₂N₅O₂ (MH⁺)403.9352, found 403.9350.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid[3-(2-amino-5-oxo-3,5-dihydro-imidazol-4-ylidene)-propyl]-amidehydrochloride (32). Using the same general procedure as used for2-amino-5-(3-amino-propylidene)-1,5-dihydro-imidazol-4 -onedihydrochloride, 0.100 g of dihydrosventrin hydrochloride 23 gave 0.048g (47%) of the title compound 32 as a tan solid ((Z)-isomerexclusively). ¹H NMR (300 MHz, CD₃OD) δ 6.84 (s, 1H), 6.16 (t, 1H, J=7.8Hz), 3.91 (s, 3H), 3.46 (t, 2H, J=6.9 Hz), 2.59 (dt, 2H, J=6.9, 14.7Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.80, 163.00, 157.19, 130.63,129.04, 119.42, 115.92, 112.67, 99.09, 39.06, 36.28, 28.73; HRMS (ESI)calcd for C₁₂H₁₄Br₂N₅O₂ (MH⁺) 417.9509, found 417.9511.

General procedure for the synthesis of tryptophan based Region C SARanalogues: Tryptamine hydrochloride (0.150 g, 0.763 mmol), the desiredappropriately substituted trichloroacetyl pyrrole (0.915 mmol), andanhydrous sodium carbonate (0.162 g, 1.53 mmol), were dissolved inanhydrous N,N-dimethylformamide (5 mL). The reaction was stirred atambient temperature for 8 h upon which it was partitioned between ethylacetate (75 mL) and water (35 mL). The organic layer was successivelywashed with water (3×20 mL), an aqueous solution of 1N HCl (2×35 mL),brine (20 mL), dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. Purification of the crude residueby column chromatography (Ethyl Acetate/Hexanes) yielded the finaltargets in the sub-library.

1H-pyrrole-2-carboxylic acid [2-(1H-indol-3-yl)-ethyl ]-amide. whitesolid (80%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.41 (s, 1H), 10.80 (s, 1H),8.11 (m, 1H), 7.58 (d, 1H, J=7.5 Hz), 7.33 (d, 1H, J=7.8 Hz), 7.16 (s,1H), 7.08 (t, 1H, J=6.6 Hz), 6.97 (t, 1H, J=7.2 Hz), 6.83 (s, 1H), 6.74(s, 1H), 6.06 (s, 1H) 3.47 (dt, 2H, J=7.2, 14.1 Hz), 2.90 (t, 2H, J=7.5Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.69, 136.27, 127.31, 126.53,122.58, 121.13, 120.95, 118.36, 118.25, 112.00, 111.40, 109.66, 108.52,39.47, 25.59; HRMS (FAB) calcd for C₁₅H₁₆N₃O (MH⁺) 254.1293, found254.1281.

4-bromo-1H-pyrrole-2-carboxylic acid [2-(1H-indol-3-yl) -ethyl]-amide.white solid (81%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.81 (s, 1H), 10.81 (s,1H), 8.23 (t, 1H, J=6.0 Hz), 7.56 (d, 1H, J=7.8 Hz), 7.33 (d, 1H, J=8.1Hz), 7.16 (s, 1H), 7.06 (t, 1H, J=6.6 Hz), 6.97 (m, 2H), 6.82 (s, 1H),3.48 (dt, 2H, J=6.9, 13.5 Hz), 2.90 (t, 2H, J=7.8 Hz); ¹³C NMR (100 MHz,DMSO-d₆) δ 159.59, 136.25, 127.26, 127.14, 122.65, 121.06, 120.95,118.31, 118.25, 111.84, 111.41, 111.26, 94.12, 25.39; HRMS (FAB) calcdfor C₁₅H₁₅BrN₃O (MH⁺) 332.0398, found 332.0388.

4,5-dibromo-1H-pyrrole-2-carboxylic acid[2-(1H-indol-3-yl)-ethyl]-amide. white solid (60%): ¹H NMR (300 MHz,DMSO-d₆) δ 12.67 (s, 1H), 10.81 (s, 1H), 8.25 (t, 1H, J=5.1 Hz), 7.57(d, 1H, J=8.1 Hz), 7.33 (d, 1H, J=8.4 Hz), 7.15 (s, 1H), 7.07 (t, 1H,J=6.9 Hz), 6.97 (t, 1H, J=7.2 Hz), 6.90 (d, 1H, J=2.7 Hz), 3.47 (dt, 2H,J=6.6, 13.2 Hz), 2.89 (t, 2H, J=7.2 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ158.87, 136.25, 128.41, 127.25, 122.70. 120.96, 118.29, 118.27, 112.42,111.76, 111.41, 104.39, 97.79, 39.57, 25.31; HRMS (EST) calcd forC₁₅H₁₄Br₂N₃O (MH⁺) 409.9498, found 409.9501.

4,5-dichloro-1H-pyrrole-2-carboxylic acid[2-(1H-indol-3-yl)-ethyl]-amide. white solid (73%): ¹H NMR (300 MHz,DMSO-d₆) δ 12.71 (s, 1H), 10.81 (s, 1H), 8.27 (m, 1H), 7.56 (d, 1H,J=7.8 Hz), 7.32 (d, 1H, J=8.1 Hz), 7.15 (s, 1H), 7.03 (t, 1H, J=6.9 Hz),6.96 (t, 1H, J=6.9 Hz), 6.86 (s, 1H), 3.47 (dt, 2H, J=6.3, 13.2 Hz),2.90 (t, 2H, J=7.5 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 159.63, 136.91,127.91, 125.80, 123.34, 121.61, 118.94, 118.91, 115.33, 112.41, 112.07,110.09, 108.54, 25.98; HRMS (FAB) calcd for C₁₅H₁₃Cl₂N₃O (M⁺) 321.0436,found 321.0429.

1-methyl-pyrrole-2-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-amide.white solid (63%): ¹H NMR (300 MHz, DMSO-d₆) δ 10.81 (s, 1H), 8.10 (t,1H, J=5.4 Hz), 7.57 (d, 1H, J=7.8 Hz), 7.33 (d, 1H, J=8.1 Hz), 7.16 (s,1H), 7.06 (t, 1H, J=7.2 Hz), 7.00 (t, 1H, J=7.8 Hz) 6.87 (s, 1H), 6.73(d, 1H, J=2.1 Hz), 6.00 (s, 1H), 3.84 (s, 3H), 3.45 (dt, 2H, J=6.9, 14.1Hz), 2.89 (t, 2H, J=7.8 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 161.31, 136.23,127.46, 127.28, 125.78, 122.57, 120.91, 118.32, 118.21, 111.98, 111.94,111.36, 106.51, 36.16, 25.49; HRMS (FAB) calcd for C₁₆H₁₈N₃O (MH⁺)268.1450, found 268.1434.

4-bromo-1-methyl-pyrrole-2-carboxylic acid[2-(1H-indol-3-yl)-ethyl]-amide. white solid (72%): ¹H NMR (300 MHz,DMSO-d₆) δ 10.81 (s, 1H), 8.20 (t, 1H, J=5.4 Hz), 7.57 (d, 1H, J=7.8Hz), 7.33 (d, 1H, J=8.1 Hz), 7.16 (d, 1H, J=1.8 Hz), 7.07 (m, 2H), 6.97(t, 1H, J=6.9 Hz), 6.80 (d, 1H, J=1.8 Hz), 3.82 (s, 3H), 3.45 (dt, 2H,J=6.9, 14.1 Hz), 2.89 (t, 2H, J=7.8 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ159.94, 136.04, 127.08, 126.63, 126.42, 122.48, 120.75, 118.12, 118.06,113.24, 111.69, 111.23, 92.74, 36.31, 25.29; HRMS (FAB) calcd forC₁₆H₁₆BrN₃O (M⁺) 345.0477, found 345.0483.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid[2-(1H-indol-3-yl)-ethyl]-amide. white solid (77%). ¹H NMR (300 MHz,DMSO-d₆) δ 10.81 (s, 1H), 8.32 (t, 1H, J=5.7 Hz), 7.55 (d, 1H, J=7.5Hz), 7.33 (d, 1H, J=8.1 Hz), 7.16 (s, 1H,) 7.06 (t, 1H, J=7.2 Hz), 7.00(t, 1H, J=7.5 Hz), 6.95 (s, 1H), 3.88 (s, 3H), 3.45 (dt, 2H, J=6.3, 13.5Hz), 2.89 (t, 2H, J=7.8 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 159.66,136.22, 128.15, 127.25, 122.67, 120.91, 118.24, 118.21, 113.78, 111.75,111.37, 110.31, 96.81, 39.66, 35.32, 25.13; HRMS (EST) calcd forC₁₆H₁₆Br₂N₃O (MH⁺) 423.9654, found 423.9655.

General procedure for the synthesis of imidazole based Region C SARanalogues (34-40): Histamine dihydrochloride 33 (0.100 g, 1.36 mmol),the desired appropriately substituted trichloroacetyl pyrrole (1.43mmol), and anhydrous sodium carbonate (0.432 g, 4.08 mmol), weredissolved in anhydrous N,N-dimethylformamide (7 mL). The reaction wasstirred at ambient temperature for 6 h upon which it was partitionedbetween ethyl acetate (75 mL) and water (35 mL). The organic layer wassuccessively washed with water (3×20 mL), sat. NaHCO₃ (2×35 mL), brine(20 mL), dried over anhydrous sodium sulfate, filtered, and concentratedunder reduced pressure. Purification of the crude residue by columnchromatography (CH₂Cl₂/Methanol 85:15) delivered the desired targets intheir free base form. Addition of concentrated HCl to a methanolicsolution (8 mL) of the free base followed by rotary evaporation affordedthe final analogues in this series as their corresponding hydrochloridesalts.

1H-pyrrole-2-carboxylic acid [2-(1H-imidazol-4-yl)-ethyl]-amidehydrochloride (34). white solid (54%): ¹H NMR (400 MHz, DMSO-d₆) δ 11.46(s, 1H), 9.01 (s, 1H), 8.23 (t, 1H, J=5.6 Hz), 7.46 (s, 1H), 6.82 (m,1H), 6.73 (m, 1H), 6.05 (dd, 1H, J=2.8, 6.0 Hz), 3.50 (dt, 2H, J=6.8,12.8 Hz), 2.87 (t, 1H, J=6.8 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 161.40,134.28, 131.82, 126.75, 122.01, 116.71, 110.80, 109.18, 37.98, 25.40;HRMS (FAB) calcd for C₁₀H₁₃N₄O (MH⁺) 205.1089, found 205.1083.

4-bromo-1H-pyrrole-2-carboxylic acid [2-(1H-imidazol-4-yl)-ethyl]-amidehydrochloride (35). white solid (40%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.80(s, 1H), 8.18 (t, 1H, J=5.4 Hz), 7.53 (s, 1H), 6.96 (s, 1H), 6.80 (s,2H), 3.41 (m, 2H), 2.70 (t, 2H, J=7.2 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ159.55, 134.72, 127.06, 121.10, 116.67, 111.28, 94.92, 38.83, 27.18;HRMS (FAB) calcd for C₁₀H₁₂BrN₄O (MH⁺) 283.0194, found 283.0198.

4,5-dibromo-1H-pyrrole-2-carboxylic acid[2-(1H-imidazol-4-yl)-ethyl]-amide hydrochloride (36). white solid(35%):. ¹H NMR (300 MHz, DMSO-d₆) δ 12.64 (br s, 1H), 8.20 (t, 1H, J=5.7Hz), 7.59 (s, 1H), 6.89 (s, 1H), 6.83 (s, 1H), 3.41 (dt, 2H, J=7.2, 13.2Hz), 2.71 (t, 2H, J=7.2 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 158.88,133.83, 131.81, 128.10, 116.27, 112.97, 104.45, 97.84, 37.82, 25.10;HRMS (FAB) calcd for C₁₀H₁₁Br₂N₄O (MH⁺) 360.9300, found 360.9295.

4,5-dichloro-1H-pyrrole-2-carboxylic acid[2-(1H-imidazol-4-yl)-ethyl]-amide hydrochloride (37). white solid(62%): ¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (s, 1H), 9.02 (s, 1H), 8.56 (t,1H, J=5.6 Hz), 7.46 (s, 1H), 6.94 (d, 1H, J=2.8 Hz), 3.50 (dt, 2H,J=6.8, 12.8 Hz), 2.89 (t, 2H, J=6.4 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ159.04, 133.53, 130.96, 124.76, 116.10, 114.73, 110.16, 107.95, 37.49,24.43; HRMS (ESI) calcd for C₁₀H₁₁Cl₂N₄O (MH⁺) 273.0304, found 273.0309.

1-methyl-pyrrole-2-carboxylic acid [2-(1H-imidazol-4-yl) -ethyl]-amidehydrochloride (38). white solid (55%): ¹H NMR (300 MHz, DMSO-d₆) δ 14.60(bs, 1H), 14.32 (bs, 1H), 9.03 (s, 1H), 8.17 (s, 1H), 7.50 (s, 1H), 6.87(s, 1H), 6.74 (s, 1H), 5.98 (d, 1H, J=2.7 Hz), 3.80 (s, 3H), 3.48 (m,2H). 2.88 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 161.32, 134.68, 127.51,125.75, 116.69. 111.98, 106.57, 38.77, 36.14, 27.17; HRMS (ESI) calcdfor C₁₁H₁₅N₄O (MH⁺) 219.1240, found 219.1245.

4-bromo-1-methyl-pyrrole-2-carboxylic acid[2-(1H-imidazol-4-yl)-ethyl]-amide hydrochloride (39). white solid(54%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.86 (br s, 1H), 8.15 (m, 1H), 7.53(s, 1H), 7.06 (d, 1H, J=1.5 Hz), 6.79 (m, 2H), 3.81 (s, 3H), 3.38 (m,2H), 2.69 (t, 1H, J=7.8 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 160.12, 134.66,126.83, 126.52, 113.36, 92.85, 38.80, 36.29, 27.00; HRMS (ESI) calcd forC₁₁H₁₄BrN₄O (MH⁺) 297.0345, found 297.0348.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid[2-(1H-imidazol-4-yl)-ethyl]-amide hydrochloride (40). white solid(60%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.85 (br s, 1H), 8.26 (t, 1H, J=5.4Hz), 7.54 (s, 1H), 6.95 (s, 1H), 6.80 (s, 1H), 3.87 (s, 3H), 3.39 (dt,2H, J=6.9, 13.8 Hz), 2.70 (t, 2H, J=7.2 Hz); ¹³C NMR (100 MHz, DMSO-d₆)δ 159.72, 133.67, 131.48, 127.78, 116.20, 114.14, 110.62, 96.88, 37.75,35.33, 24.71; HRMS (FAB) calcd for C₁₁H₁₃Br₂N₄O₂ (MH⁺) 374.9456, found374.9458.

4-(3-amino-propyl)-1,3-dihydro-imidazole-2-thione hydrochloride (42). Toan Erlenmeyer flask was prepared a solution of L-ornithine methyl esterhydrochloride (10.50 g, 47.9 mmol) in water (125 mL). The solution wascooled to 5° C. and pH adjusted to a value of 1.5 with concentrated HCl.While being careful to maintain the above stated temperature and pH, 5%Na(Hg) (250 g) was added slowly to the solution over a time period of 35min. After the addition was complete and bubbling had calmed the Hg wasdecanted from the solution. The remaining aqueous portion was drainedinto a separate flask where potassium thiocyanate (14.0 g, 144 mmol) andwater (75 mL) was added. The pH of solution was adjusted to a value of4.30 and the flask was then heated at 95° C. while open to theatmosphere for 1.5 h. After cooling to room temperature, ethanol (75 mL)was added and the reaction was evaporated to dryness. The residue wastaken up in methanol and filtered to remove NaCl. After all of the NaClhad been removed the crude residue was purified by column chromatography(CH₂Cl/MeOH sat. with NH₃ 80:20) to afford the desired compound in itsfree base form. Addition of concentrated hydrochloric acid to a methanolsolution (50 mL) of the free base followed by evaporation to drynessgave 4.51 g (48%) of the title compound 42 as a tan solid: ¹H NMR (300MHz, DMSO-d₆) δ 11.92 (s, 1H), 11.69 (s, 1H), 7.77 (s, 2H), 6.58 (s,1H), 2.73 (m, 2H), 2.42 (t, 2H, J=6.6 Hz), 1.76 (tt, 2H, J=7.5, 13.8Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.23, 128.03, 111.69, 38.08, 25.75,21.27; HRMS (FAB) calcd for C₆H₁₂N₃S (MN⁺) 158.0752, found 158.0743.

General procedure for the synthesis of 2-thioimadazolone Region C SARanalogues (43-49): 4-(3-amino-propyl)-1,3-dihydro-imidazole-2-thionehydrochloride 42 (0.150 g, 0.774 mmol), the desired appropriatelysubstituted trichloroacetyl pyrrole (0.852 mmol), and anhydrous sodiumcarbonate (0.246 g, 2.32 mmol), were dissolved in anhydrousN,N-dimethylformamide (5 mL). The reaction was stirred at ambienttemperature for 12 h upon which it was partitioned between ethyl acetate(75 mL) and water (35 mL). The organic layer was successively washedwith water (3×20 mL), a 1N aqueous solution of HCl (2×35 mL), brine (20mL), dried over anhydrous sodium sulfate, filtered, and concentratedunder reduced pressure. Purification of the crude residue by columnchromatography (CH₂Cl₂/Methanol) afforded the final analogues in thisseries.

1H-pyrrole-2-carboxylic acid[3-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)-propyl]-amide (43). paleyellow solid (53%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.40 (s, 1H), 8.20 (t,1H, J=5.7 Hz), 6.97 (s, 1H), 6.82 (m, 1H), 6.75 (m, 1H), 6.06 (dd, 1H,J=2.1, 5.4 Hz), 3.23 (dt, 2H, J=6.6, 12.9 Hz), 2.52 (m, 2H), 1.75 (tt,2H, J=7.2, 14.7 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 160.80, 160.71, 130.67,126.31, 121.14, 113.24, 109.77, 108.47, 37.67, 28.32, 22.01; HRMS (FAB)calcd for C₁₁H₁₅N₄OS (MH⁺) 251.0967, found 251.0961.

4-bromo-1H-pyrrole-2-carboxylic acid[3-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)-propyl]-amide (44). paleyellow solid (52%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.87 (s, 1H), 11.81 (s,1H), 11.65 (s, 1H), 8.10 (t, 1H, J=5.1 Hz), 6.96 (s, 1H), 6.83 (s, 1H),6.57 (s, 1H), 3.18 (dt, 2H, J=6.3, 12.3 Hz), 2.37 (t, 2H, J=7.5 Hz),1.71 (tt, 2H, J=6.9, 13.8 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 160.35,160.30, 130.05, 127.63, 121.74, 112.76, 112.04, 95.56, 38.43 28.77,22.52; HRMS (ESI) calcd for C₁₁H₁₄BrN₄OS (MH⁺) 329.0066, found 329.0062.

4,5-dibromo-1H-pyrrole-2-carboxylic acid[3-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)-propyl]-amide (45). whitesolid (41%): ¹H NMR (300 MHz, DMSO-d₆) δ 12.68 (s, 1H), 11.86 (s, 1H),11.65 (s, 1H), 8.13 (t, 1H, J=5.1 Hz), 6.91 (d, 1H, J=2.7 Hz), 6.57 (s,1H), 3.17 (m, 2H), 2.36 (t, 2H, J=6.9 Hz), 1.70 (tt, 2H, J=6.9, 13.8Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 160.04, 158.91, 128.85, 128.02, 112.45,111.30, 104.43, 97.76, 37.78, 27.95, 21.72; HRMS (ESI) calcd forC₁₁H₁₃Br₂N₄OS (MH⁺) 406.9171, found 406.9174.

4,5-dichloro-1H-pyrrole-2-carboxylic acid[3-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)-propyl]-amide (46). yellowsolid (65%): ¹H NMR (300 MHz, DMSO-d₆) δ 12.71 (s, 1H), 11.86 (s, 1H),11.65 (s, 1H), 8.15 (m, 1H), 6.86 (s, 1H), 6.56 (s, 1H), 3.17 (m, 2H),2.36 (t, 2H, J=7.5 Hz), 1.71 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ160.73, 159.70, 129.53, 125.60, 115.40, 111.97, 110.16, 108.55, 38.47,28.62, 22.40; HRMS (FAB) calcd for C₁₁H₁₂Cl₂N₄OS (M⁺) 318.0109, found318.0099.

1-methyl-pyrrole-2-carboxylic acid[3-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)-propyl]-amide (47). paleyellow solid (57%): ¹H NMR (300 MHz, DMSO-d₆) δ 7.99 (m, 1H), 6.99 (s,1H), 6.86 (s, 1H), 6.74 (m, 1H), 5.98 (m, 1H), 3.20 (dt, 2H, J=6.0, 12.3Hz), 2.54 (t, 2H, J=7.2 Hz), 1.77 (tt, 2H, J=6.9, 14.1 Hz); ¹³C NMR (75MHz, DMSO-d₆) δ 161.38, 160.95, 127.51, 125.64, 115.10, 112.09, 106.50,97.60, 37.71, 36.11, 28.32, 22.15; HRMS (ESI) calcd for C₁₂H₁₇N₄OS (MH⁺)265.1118, found 265.1120.

4-bromo-1-methyl-pyrrole-2-carboxylic acid[3-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)-propyl]-amide (48). yellowsolid (54%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.86 (s, 1H), 11.64 (s, 1H),8.07 (m, 1H), 7.07 (s, 1H), 6.81 (s, 1H), 6.56 (s, 1H), 3.80 (s, 3H),3.13 (dt, 2H, J=6.3, 12.9 Hz), 2.36 (t, 2H, J=7.8 Hz), 1.69 (m, 2H); ¹³CNMR (75 MHz, DMSO-d₆) δ 160.25, 160.00, 128.95, 126.85, 126.45, 113.45,111.34, 92.87, 37.70, 36.30, 27.96, 21.78; HRMS (ESI) calcd forC₁₂H₁₆BrN₄OS (MH⁺) 343.0222, found 343.0223.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid[3-(2-thioxo-2,3-dihydro-1H-imidazol-4-yl)-propyl]-amide (49). whitesolid (71%): ¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (s, 1H), 11.64 (s, 1H),8.19 (t, 1H, J=5.2 Hz), 6.97 (s, 1H), 6.55 (s, 1H), 3.86 (s, 3H), 3.15(dt, 2H, J=6.4, 12.4 Hz), 2.36 (t, 2H, J=7.2 Hz), 1.70 (tt, 2H, J=7.2,14.0 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 160.00, 159.76, 128.89, 128.10,113.84, 111.33, 110.42, 96.84, 37.86, 35.35, 27.85, 21.77; HRMS (ESI)calcd for C₁₂H₁₅Br₂N₄OS (MH⁺) 420.9328, found 420.9327.

1-bromo-5-phthalimido-2-pentanone. 4-phthalimidobutanoic acid 50 (4.64g, 19.9 mmol) was dissolved in CH₂Cl₂ (100 mL) at 0° C. and a catalyticamount of DMF was added. To this solution was added oxalyl chloride (5.2mL, 59.6 mmol) drop-wise and the solution was then warned to roomtemperature. After 1 h, the solvent and excess oxalyl chloride wereremoved under reduced pressure. The resulting solid was dissolved intoCH₂Cl₂ (10 mL) and added slowly to a 0° C. solution of CH₂N₂ (˜60 mmolgenerated from Diazald® diazomethane precursor/KOH) in Et₂O (170 mL).This solution was stirred at 0° C. for 1.5 h at which time the reactionwas quenched with the drop-wise addition of 48% HBr (7.0 mL). Thereaction mixture was diluted with CH₂Cl₂ (50 mL) and immediately washedwith sat. NaHCO₃, brine, dried (MgSO₄), filtered and concentrated. Theresulting white solid was filtered and washed with Et₂O (100 mL) toobtain the title compound (4.77 g, 84%) as a fine white powder: ¹H NMR(300 MHz, DMSO-d₆) δ 7.85 (m, 4H), 4.32 (s, 2H), 3.57 (t, 2H, J=6.9 Hz),2.65 (t, 2H, J=6.9 Hz), 1.82 (quint., 2H, J=6.9 Hz); ¹³C NMR (75 MHz,DMSO-d₆) δ 200.94, 168.05, 134.34, 131.68, 123.01, 36.94, 36.64, 36.37,22.30; HRMS (ESI) calcd for C₁₃H₁₃BrNO₃ (MH⁺) 310.0073, found 310.0072.

2-amino-4-(3-phthalimidopropyl)thiazole (51).1-bromo-5-phthalimido-2-pentanone (0.500 g, 1.61 mmol) was dissolved inDMF (3.5 mL) at 0° C. and thiourea (0.135 g, 1.77 mmol) was addeddrop-wise as a solution in DMF (0.50 mL). The solution was allowed towarm to room temperature and stirring was continued for 2 h at whichtime the DMF was removed under reduced pressure and the resulting slurrywas made alkaline with 10% K₂CO₃ (100 ML). The aqueous solution was thenextracted with EtOAc (3×40 mL) and the organic layer was washed withbrine (50 mL), dried (Na₂SO₄), filtered and concentrated to obtain 51(448 mg, 97%) as a fine white powder in its freebase form: ¹H NMR (300MHz, DMSO-d₆) δ 7.85 (m, 4H), 6.78 (s, 2H), 6.13 (s, 1H), 3.56 (t, 2H,J=6.9 Hz), 2.42 (t, 2H, J=7.5 Hz), 1.88 (quint., 2H, J=7.5 Hz); ¹³C NMR(75 MHz, DMSO-d₆) δ 168.01, 167.87, 151.20, 134.25, 131.61, 122.89,100.17, 37.27, 28.70, 27.09; HRMS (ESI) calcd for C₁₄H₁₄N₃O₂S (MH⁺)288.0801, found 288.0799.

2-amino-4-(3-aminopropyl)thiazole dihydrochloride.2-amino-4-(3-phthalimidopropyl)thiazole (51) (0.300 g, 1.04 mmol) wasdissolved in MeOH (4.5 mL) and N₂H₄ (0.10 mL, 3.20 mmol) was addeddrop-wise to the stirring solution. The solution was stirred at roomtemperature for 1 h, warmed to 55° C. for 0.5 h and then cooled to roomtemperature. The slurry was filtered and the filtrate concentrated underreduced pressure. The resulting residue was purified by flash columnchromatography (50-100% MeOH/CH₂Cl₂; followed by 5-7% TEA/MeOH) toobtain the corresponding freebase (0.148 g, 90%) as a fine white powder.Addition of concentrated HCl to a cold methanolic solution (8 mL) of thefreebase followed by evaporation under reduced pressure delivered thetitle compound as its dihydrochloride salt. ¹H NMR (300 MHz, DMSO-d₆) δ9.07 (bs, 2H), 8.06 (bs, 3H), 6.57 (s, 1H), 2.78 (m, 2H), 2.62 (t, 2H,J=7.2 Hz), 1.87 (quint., 2H, J=7.2 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ169.90, 139.21, 102.13, 37.68, 25.10, 24.47; HRMS (ESI) calcd forC₆H₁₂N₃S (MH⁺) 158.0746, found 158.0745.

General procedure for the synthesis of 2-AT Region C SAR analogues:2-amino-4-(3-aminopropyl)thiazole (0.200 mmol), the appropriatelysubstituted trichloroacetyl pyrrole (0.210 mmol) and anhydrous potassiumcarbonate (0.300 mmol) were dissolved in anhydrous N,N-dimethylformamide(1.5 mL) and allowed to stir for 16 h at room temperature. The mixturewas then concentrated under reduced pressure and the resulting residuewas dissolved in EtOAc (40 mL) and washed with H₂O (3×20 mL) and brine(20 mL), dried (Na₂SO₄), filtered and concentrated. The crude residuewas purified by flash column chromatography (30-100% EtOAc/Hexanes;followed by 5-10% MeOH/EtOAc) to obtain pure product. Addition ofconcentrated HCl to a methanolic solution (5 mL) of the freebasefollowed by concentration under reduced pressure afforded the requisiteanalogues for this series as their hydrochloride salts.

1H-pyrrole-2-carboxylic acid [3-(2-amino-thiazol-4-yl) -propyl]-amidehydrochloride. tan solid (64%): ¹H NMR (300 MHz, DMSO-d₆) 11.51 (s, 1H),9.26 (s, 2H), 8.21 (s, 1H), 6.84 (s, 1H), 6.79 (s, 1H), 6.59 (s, 1H),6.07 (d, 1H, J=2.7 Hz), 3.24 (q, 2H, J=5.7 Hz), 2.57 (t, 2H, J=7.2 Hz),1.79 (quint., 2H, J=7.2 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 169.90, 160.68,139.79, 126.28, 121.02, 110.02, 108.34, 101.61, 37.49, 27.59, 24.93;HRMS (ESI) calcd for C₁₁H₁₅N₄OS (MH⁺) 251.0961, found 251.0960.

4,5-dibromo-1H-pyrrole-2-carboxylic acid [3-(2-amino-thiazol-4-yl)-propyl]-amide hydrochloride. tan solid (56%): ¹H NMR (400 MHz,DMSO-d₆) 12.76 (s, 1H), 9.19 (s, 2H), 8.36 (t, 1H, J=5.6 Hz), 6.96 (d,1H, J=2.0 Hz), 6.58 (s, 1H), 3.23 (q, 2H, J=7.2 Hz), 2.56 (t, 2H, J=7.2Hz), 1.78 (quint., 2H, J=7.2 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 169.91,158.87, 139.93, 128.23, 112.81, 104.20, 101.70, 97.76, 37.67, 27.35,25.00; HRMS (ESI) calcd for C₁₁H₁₃Br₂N₄OS (MH⁺) 406.9171, found406.9165.

1-methyl-pyrrole-2-carboxylic acid [3-(2-amino-thiazol-4-yl)-propyl]-amide hydrochloride. tan solid (56%): ¹H NMR (300 MHz, DMSO-d₆)9.19 (br s, 2H), 8.08 (m, 2H), 6.88 (t, 1H, J=2.1 Hz), 6.78 (dd, 1H,J=3.9 & 2.1 Hz), 6.57 (s, 1H), 5.99 (dd, 1H, J=3.9 & 2.7 Hz), 3.82 (s,3H), 3.20 (q, 2H, J=7.2 Hz), 2.55 (t, 2H, J=7.2 Hz), 1.78 (quint, 2H,J=7.2 Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 169.83, 161.37, 139.93, 127.40,125.54, 112.04, 106.40, 101.54, 37.44, 35.94, 27.52 24.98; HRMS (ESI)calcd for C₁₂H₁₇N₄OS (MH⁺) 265.1118, found 265.1117.

4,5-dibromo-1-methyl-pyrrole-2-carboxylic acid [3-(2-amino-thiazol-4-yl)-propyl]-amide hydrochloride. tan solid (62%): ¹H NMR (400MHz, DMSO-d₆) 9.02 (br s, 2H), 8.29 (n, 1H), 7.02 (s, 1H), 6.55 (s, 1H),3.87 (s, 3H), 3.19 (q, 2H, J=6.8 Hz), 2.53 (m, 2H), 1.77 (t, 2H, J=6.8Hz); ¹³C NMR (75 MHz, DMSO-d₆) δ 169.92, 159.83, 140.23, 128.03, 114.03,110.40, 101.71, 96.86, 37.78, 35.34, 27.31, 25.14; HRMS (ESI) calcd forC₁₂H₁₅Br₂N₄OS (MH⁺) 420.9328, found 420.9321.

EXAMPLE 6

Inhibition and dispersion of proteobacterial biofilms withdihydrooroidin derivatives. A dihydrooroidin library was assembled asdescribed above by solution-based synthetic methods, and each member ofthe library was fully characterized (¹H NMR, ¹³C NMR, HRMS). Theanalogue library was subsequently screened in a 96-well format using acrystal violet reporter assay to assess each analogue's ability toinhibit the formation of P. aerutginosa biofilms. From this initialscreen, the derivative dihydrosventrin (DHS) (FIG. 20) was discovered tobe the most potent member of the library and was further evaluated.

Follow-up dose response experiments revealed that DHS had IC₅₀ values of51 μM against PAO1 and 111 μM against PA14 (Table 5), indicating thatDHS was approximately 2-fold more active than both TAGE and CAGE. It isnoteworthy that DHS displayed much greater activity than both oroidin 5(IC₅₀=190 μM PAO1, IC₅₀=166 μM PA14) and its unsaturated parent sventrin6 (IC₅₀=75 μM PAO1, IC₅₀=115 μM PA14). Dihydroorodin 4 showed onlymarginal activity (<70%) at 500 μM. Comparison of both growth curves andcolony counts for PAO1 and PA14 grown in the absence or presence of DHS(7), oroidin (5), and sventrin (6) indicated that the inhibition ofbiofilm formation was not due to a bactericidal effect (not shown).

Given that DHS displayed exceptional activity in inhibiting theformation of PAO1 and PA14 biofilms, it was determined whether it wouldalso inhibit the formation of a mucoid variant of P. aeruginosa. After aCF patient is colonized by P. aeruginosa, the bacterium undergoes aphenotypic shift from a non-mucoid to a mucoid form. J. Govan and V.Deretic, Microbiol. Rev. 1996, 60, (3), 539-74. D. Ramsey and D.Wozniak, Mol. Microbial 2005, 56, (2), 309-22. Numerous studies havecorrelated the appearance of mucoid P. aeruginosa with a decline in thepulmonary clinical status of CF patients. J. Govan and V. Deretic,Microbiol. Rev. 1996, 60, (3), 539-74. R. Fick, et al., Semin. Respir.Infect. 1992, 7, (3), 168-78. R. Doggett et al., Lancet 1971, 1, (7692),236-7. PDO300 (K. Mathee, et al., Microbiology 1999, 145 (Pt 6),1349-57.) was employed to assay if DHS would inhibit the formation ofmucoid biofilms. PDO300 is a well-characterized mucoid strain of P.aeruginosa that is genotypically identical to PAO1 except for the mucAmutation that converts the bacterium to the mucoid phenotype. K. Matheeet al., Microbiology 1999, 145 (Pt 6), 1349-57. It was determined thatDHS has an IC₅₀ of 115 μM against PDO300 (Table 5). Growth curves againindicated that DHS lacked significant bactericidal activity againstPDO300.

TABLE 5 IC₅₀ Values for DHS Across Selected Proteobacteria^(a)Proteobacterial Strain IC₅₀ value for DHS (μM) P. aeruginosa (PAO1) 51P. aeruginosa (PA14) 111 P. aeruginosa (PDO300) 115 A. baumannii (Actb)110 B. bronchiseptica (RB50) 238 ^(a)All assays performed in triplicate.

The ability of DHS to inhibit the formation of Acinetobacter baumanniibiofilms was also assessed. A. baumannii is an opportunisticγ-proteobacterium that has become a severe threat over the last decadedue to its multi-drug resistance. M. Falagas and E. Karveli, Clin.Microbiol. Infect. 2007, 13, (2), 117-119. Approximately 25% of allhospital swabs are positive for A. baumannii. D. Forster and F.Daschner, Eur. J. Clin. Microbiol. Infect. Dis. 1998, 17, (2), 73-77. A.baumannii survives for weeks on dry surfaces due to its ability to formrobust biofilms. A. Tomaras, et al., Microbiology-Sgm 2003, 149,3473-3484. Clearly, this is a serious impediment to control strategies,and small molecules that inhibit A. baumannii biofilms may beparticularly valuable for A. baumannii remediation efforts. There arecurrently no known small molecules documented that inhibit A. baumanniibiofilm formation. DHS was slightly more potent against A. baumannii asit was against PA14, revealing an IC₅₀ value of 110 μM (Table 5). Bothgrowth curves and colony counts of A. baumannii grown in the absence orpresence of DHS indicated this compound has no microbiocidal effects.

It was next determined if DHS had the ability to inhibit the formationof biofilms across bacterial order. The Bordetella bronchiseptica strainRB50, a β-proteobacterium, was chosen for evaluation. Bacteria of thegenus Bordetellae are frequently isolated from mammalian respiratorytracts. P. Cotter and J. Miller, Mol. Microbiol. 1997, 24, (4), 671-685.B. bronchiseptica shares many of the same virulence factors asBordetella pertussis, a β-proteobacterium that causes whooping cough andis responsible for 300,000 fatalities per year. N. Crowcroft and J.Britto, Brit. Med. J. 2002, 324, (7353), 1537-1538. DHS was found tohave an IC₅₀ of 238 μM against RB50 (Table 5).

From a clinical perspective, the ability to disperse an establishedbiofilm can be an important consideration, because a physician istypically faced with treating an established or chronic biofilminfection. DHS was assessed for its ability to disperse existingbiofilms of P. aeruginosa, A. baumannii, and B. bronchiseptica. Eachbacterial strain was allowed to form biofilms for 24 hours in theabsence of compound. At the end of 24 hours, the media and planktonicbacteria were removed and the remaining biofilm was treated either withmedia alone or media containing DHS. DHS was able to successfullydisperse each biofilm throughout a range of concentrations (FIG. 21).

In conclusion, a simple derivative of a marine alkaloid has beenidentified that is active in inhibiting and dispersing proteobacterialbioflims. DHS inhibits and disperses the formation of bacterial biofilmsacross bacterial order. Given this activity, DHS and related compoundsare useful for the development of therapeutics directed towardcontrolling and eliminating biofilm infections.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A compound of Formula (X)(I)(a)(1):

wherein: n is 2, 3 or 4, saturated or unsaturated; and R⁶ is selectedfrom the group consisting of H, alkyl, alkenyl and alkynyl; or apharmaceutically acceptable salt or prodrug thereof.
 2. The compound ofclaim 1, wherein R⁶ is an alkyl having from 5 to 20 carbon atoms.
 3. Thecompound of claim 1, wherein n is
 3. 4. The compound of claim 1, whereinsaid compound represented by Formula (X)(I)(a)(1) is a compound ofFormula (X)(I)(a)(1)(A):

or a pharmaceutically acceptable salt or prodrug thereof.
 5. Acomposition comprising the compound of claim 1 in a pharmaceuticallyacceptable carrier.
 6. A composition comprising a compound of Formula(X)(I)(a)(1):

wherein: n is 2, 3 or 4, saturated or unsaturated; and R⁶ is selectedfrom the group consisting of H, alkyl, alkenyl and alkynyl; or apharmaceutically acceptable salt or prodrug thereof; covalently coupledto a substrate.
 7. The composition of claim 6, wherein said substratecomprises a polymeric material.
 8. The composition of claim 6, whereinsaid substrate comprises a solid support.
 9. The composition of claim 6,wherein said substrate is selected from the group consisting of adrainpipe, glaze ceramic, porcelain, glass, metal, wood, chrome,plastic, vinyl, and formica.
 10. The composition of claim 6, wherein thesubstrate is selected from the group consisting of shower curtains orliners, upholstery, laundry, and carpeting.
 11. The composition of claim6, wherein said substrate is a ship hull or portion thereof.
 12. Thecomposition of claim 6, wherein the substrate is a food contact surface.13. The compound of claim 2, wherein R⁶ is a branched alkyl having from5 to 20 carbon atoms.
 14. The composition of claim 6, wherein R⁶ is analkyl having from 5 to 20 carbon atoms.
 15. The composition of claim 6,wherein n is 3.